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Chapter 4Laboratory Safety Procedures

This chapter gives general guidance for working safely with chemicals in laborato-ries. While it is thorough, it is not meant to be a complete safety manual. Eachlaboratory should have an additional safety references. We recommend:

w Safety in Academic Chemistry Laboratories, Fifth Edition, a publication of theAmerican Chemical Society, 1990. Call 1-800-227-5558. One copy is free uponrequest, multiple academic copies are available for a nominal fee.

w Prudent Practices for Handling Hazardous Chemicals in Laboratories, NationalResearch Council, National Academy Press, Washington DC, 1981.

w Prudent Practices in the Laboratory, National Research Council, NationalAcademy Press, Washington DC, 1995.

Using this section in conjunction with other safety references will help you maintaina safe laboratory. It will also help you comply with the U.S. Occupational Safetyand Health Administration's (OSHA) standard for Occupational Exposure to Hazard-ous Chemicals in Laboratories (see Appendix B for details of the OSHA LabStandard). This chapter has information which will help you prepare a ChemicalHygiene Plan (see Appendix C) for your laboratory.

4.1 Overview of Safety ProceduresBy its nature, research is inherently hazardous. The use of new techniques andprocesses to investigate a hypothesis creates inherent hazards and risks. In 1890, thechemist, August KekulP, allegedly told a student, "If you want to become a chemist,you have to ruin your health. Who does not ruin his health by his studies, nowadayswill not get anywhere in Chemistry."

While today, the attitude of "safety first" permeates the research community,working safely not just something you fall into, it consists of systematically integrat-ing safety procedures into your work with hazardous materials. In Chapter 2 wediscussed terminology and concepts for working safely. Here we will review someof the general procedures designed to insure your safety.

4.1.a Responsibilities for SafetyFaculty, staff, students and the Safety Department must work together to make theUniversity a safe place to learn and to conduct research.

Employees and StudentsYou are the person most responsible for the safe use of chemicals in your lab.Research safety requires careful planning to ensure that experiments are designed ina safe manner. Chapters 1 and 2 have described your safety network and considera-tions for understanding the risks of laboratory hazards. Your responsibility for thesafety use of laboratory chemicals and equipment means:w Review and following your lab's Chemical Hygiene Plan and the safety proce-

dures of your department and the University.w Read the Material Safety Data Sheet (MSDS) for each chemical you will use prior

to first use and discussing with your principal investigator or supervisor possible

Laboratory Safety Guide

Laboratory safety iseveryone'sresponsibility.

Before working withany chemical, readthe container labelsand MSDS.

safety precautions for any laboratory-synthesized, newly recovered, rare, or exoticchemicals.

w Understand the proper use of personal protective equipment, engineering controls(e.g., fume hoods), and administrative controls.

w Avoid overexposure to hazardous chemicals, and immediately reporting anyactual or probable overexposure to your supervisor. If you experience immediateor delayed symptoms of chemical exposures, seek medical evaluation.

w Report all spills, accidents and injuries to your supervisor immediately.w Dispose hazardous waste according to University procedures.w Do not perform any procedure unless you are confident of its safety. If you are

concerned about exposure, stop and ask for help. Persons who work with labora-tory chemicals should not be afraid to ask questions, this is a learning institution.

Faculty, Instructors and Lab ManagersIf you supervise or proctor others who use chemicals, you are responsible for theirtraining and safety. To insure your workers and students will remain safe:w Check to see if necessary and appropriate safety equipment and supplies are

present and in good working condition.w Review laboratory work practices insuring safety procedures are followed.w Comply with the OSHA Laboratory Standard (see Appendix B) and have a

Chemical Hygiene Plan (see Appendix C) that is reviewed annually.w Submit accident reports to the Worker's Compensation Office immediately.w Insure new students and staff have appropriate training. Appendix G contains a

safety training outline.w Visit the Safety Department web site (http://www.fpm.wisc.edu/safety) and insure

workers attend appropriate training.w Maintain a file or binder of Material Safety Data Sheets (MSDS) for all the

chemicals in your lab. State purchasing rules require chemical suppliers toinclude an MSDS in the container package. Call the supplier, your purchasingagent or the Safety Department if you have difficulty obtaining an MSDS. Alter-natively, access to MSDSs can be provided electronically through the Internet.

w Discuss safety issues at lab group meeting and integrate safety into procedures toenhance your personnel's learning experience.

Departments, Centers, Schools and Collegesw Establish safety committees which discuss issues and safety solutions.w Promulgate a departmental Chemical Hygiene Plan to facilitate faculty plans and

establish a baseline.w Insure new faculty are made aware of both their responsibilities for safety and

assets (cf., Chapter 1) to assist them in establishing a new lab.

Safety DepartmentWhile the basic regulations published by federal agencies change little, minorchanges and interpretations are frequent. The Safety Department:w Provides information and advice on the safe use and disposal of chemicals.w Audits laboratory groups for general safety concerns.w Verifies University compliance with the various federal, state and local environ-

mental health and safety laws.w Facilitates waste minimization by redistributing surplus chemicals.w Provides a central source of MSDSs for chemicals used on campus.

76 Laboratory Safety Procedures

University of Wisconsin-Madison Safety Department (608) 262-8769

w Inspects campus safety showers, eye wash stations, fume hoods and fire extin-guishers annually to ensure their proper operation.

w Coordinates campus chemical emergency response with the Madison Fire Depart-ment's Hazardous Incident Response Team.

w Minimizes the University's liability by ensuring that hazardous waste is disposedof safely and properly.

The Safety Department is also a valuable training resource to help you and your staffbetter understand laboratory hazards and to control them. Some of the ways Safetyassists in training your workers:

w Conduct a "Working Safely with Chemicals" class weekly. This class, usuallyheld at Union South, provides the basic information necessary to satisfy theOSHA training requirements. Personnel who attend the class and successfullycomplete the quiz receive a certificate which can be used by research labsthroughout campus to satisfy the OSHA training documentation requirement.

w Provide training to targeted groups upon request. This training can either be acustomized program (e.g., for incoming graduate students, for "gifted" studentprograms, etc.), or a routine program designed to catch all your students in asingle setting and it can address the safety concerns of your department, buildingor research group and/or include the full "Working Safety with Chemicals" class.Safety personnel can also speak at a group or departmental meeting.

w Lend safety videos from our library of safety related video tapes. Other UWSystem schools may also have safety videos. Call Safety for a list.

w Publish the Chemical Safety and Disposal Guide. Appendix G has a suggestedoutline for lab worker safety training. Each section of the Guide has reviewquestions that can serve as a "Safety Quiz" to get your personnel thinking aboutlaboratory safety issues.

w Publish a CARP Spectrum newsletter which addresses safety issues affectingresearch labs on campus.

How One Lab Ensures SafetyOne lab involves each group member in keeping their lab safe. Each ofthese duties are rotated among lab workers so they can appreciate theimportance of each task.

1. Keeping an updated chemical inventory.2. Keeping all common use areas clean and orderly.3. Keeping Material Safety Data Sheet files current.4. Disposing of wastes as they are generated.5. Keeping abreast of current issues and new developments in laboratory

safety, by finding and distributing information.6. Surveying the lab for possible safety problems (see Appendix E).

Annually, the lab members jointly review and update their laboratory's

4.1.b Safe Laboratory PracticesStep back and look at your laboratory as if you were seeing it for the first time. Thisis the view that new faculty, staff, students and visitors to your lab see. Does it looksafe? Is it neat and orderly? Are people taking safety precautions? Is there a strongodor of volatile chemicals? Does it look like chemicals are stored safely? Can you

Laboratory Safety Procedures 77


Start your semesterby attendingSafety's weeklyWorking Safely withChemicals trainingclass.

see ways to make your lab safer? Better yet, conduct a more systematic survey ofyour laboratory's safety practices by using one of the survey forms in Appendix E.

Annex 2-2 to Chapter 2 presented a quick guide to risk assessment for hazardouschemicals. When you plan laboratory procedures, use that guide and these basicprinciples to help assure safety:ü Know about the chemicals and hazards associated with your lab. Know their

potential flammability, reactivity, corrosivity and toxicity. Know how to readand interpret MSDSs.

ü Know what to do in different emergency situations.ü Avoid working alone in a laboratory.ü Don't underestimate risks, assume any mixture will be more hazardous than its

most hazardous component and that all substances of unknown toxicity are toxic.ü Minimize all chemical exposures. Few laboratory chemicals are without hazards.

Use precautions when handling all laboratory chemicals. Wear personal protec-tive equipment appropriate to the work. Do not wear contact lenses aroundchemicals, fumes, dust particles or other hazardous materials.

ü Use extreme care when working with needles, blades and glass.ü Do not eat, drink, apply makeup or use tobacco products in the laboratory. Do

not mouth pipet. Do not use ice from a laboratory ice machine for humanconsumption. Dedicate microwave ovens and other heating devices exclusivelyfor food or for laboratory operations. Ensure that ovens are clearly labeled toindicate their function.

ü Provide adequate ventilation. The best way to prevent exposure to airbornesubstances is to vent them away from you. This is accomplished by using fumehoods and other ventilation devices. Avoid using dry ice in enclosed areas, it canproduce elevated carbon dioxide levels. Dry ice mixed with isopropanol orethanol may cause frostbite. Avoid producing aerosols.

ü Protect unattended operations from utility failures and other potential problemsthat could lead to overheating or other hazardous events.

ü Clean contaminated equipment and spills immediately. Avoid contaminatingequipment with mercury and clean mercury spills immediately. Use non-mercurythermometers when appropriate.

ü Keep hallways, corridors and exit ways clear. Do not locate laboratory equip-ment or supplies in these areas.

4.1.c Laboratory Safety SurveysHow do you know if your laboratory is a safe place? One way to find out is to audityour laboratory for safety practices and facilities. This can be easily accomplishedby using the Laboratory Safety Survey in Appendix E.

Other audit / survey resources are available. The Madison Fire Departmentinspects laboratories on campus to check fire suppression systems, alarms and fireextinguishers. They help us prevent fires by looking for problems, like improper orexcess storage of flammable liquids. The Safety Department is always available tohelp you with safety problems and it annually performs safety surveys of laborato-ries. Upon request, Safety can also review a specific area or practice or perform anoverall survey of your building. The Safety Department audit process involves:w Discussing your concerns and safety issues.w Touring your areas to assess the risks to health, property or the environment.



Keeping an orderly,well maintained labcan eliminate manylab safetyproblems.

Never underesti-mate the hazardsassociated with alab. If you areunsure about whatyou are doing, getassistance.

w Comparing your practices and facilities to current and accepted safety standards.w Discussing solutions, including employee training, personal protective equipment,

improved ventilation or further testing and evaluation. When appropriate, we willwork with you to find funds to improve facilities.

4.1.d Good Housekeeping Facilitates SafetyGenerally speaking, a clean, orderly lab is a safe lab. Good housekeeping can lowerthe number of lab accidents and reduce the risk and consequences of a fire. It canalso increase your working space. For a safe and efficient laboratory:ü Keep passageways to exits clear.ü Do not block areas around safety showers, fire extinguishers, fire blankets and

electrical (on/off) controls.ü Do not store chemical containers on the floor where they may be broken or

become a trip hazard for workers.ü Return chemical containers to their proper storage location after use.ü Do not use floors, stairways or hallways as storage areas.ü Keep balances, hoods, centrifuges, incubators, refrigerators, ovens and other

common-use items clean and neat for the next user.

4.1.e Laboratory and Personal HygieneGood personal hygiene will help minimize exposure to hazard chemicals. Labora-tory hygiene consists of practices to avoid accidental or inadvertent exposure tolaboratory chemicals. If there is a small spill of a fine powder, a careless coworkercan quickly spread contamination throughout a laboratory. Even small exposuresfrom some compounds may result in harmful effects for people who work in labora-tories every day. Your goal should be no skin contact with laboratory chemicals.Unlike radioactive materials, chemical contamination is difficult to detect. Whendyes have been spilled in University buildings, it is unnerving to see how widely thecontamination has spread. To keep yourself and others safe from accidentalcontamination:ü Do not touch things that are used by non-gloved hands (e.g., telephone, door

knobs, etc.) if you are wearing gloves that have touched chemicals. Gloves canbe washed with soap and water in many cases if removal is not convenient.

ü Wash your hands in the lab frequently, especially after lab work and beforeeating, drinking, applying makeup or leaving the area.

ü Routinely wash doorknobs, telephones, keyboards and desks.ü Never use mouth suction to fill a pipet. Use a pipet bulb or other mechanical

pipet filling device.ü Don't keep food in refrigerators used to store chemical, radioactive or biological

experiments. Do not use laboratory equipment to serve or store food or drinks.ü Never eat, drink or smoke in labs (smoking isn't allowed in any UW building).ü Do not wear contact lenses near chemicals, especially corrosives or volatile

solvents.ü Never allow a laboratory chemical to touch your skin; use gloves and wear a lab

coat. Remove contaminated clothing immediately and do not use the clothingagain until it has been properly decontaminated.

ü Don't immerse your fingers or hand in liquids; use tongs or a tool.ü Do not sniff or taste chemicals.



Wall-mountedshelves havedetached and fallenonto desks andother worksurfaces. Theseshelves wereheavily loaded,exceeding the loadcapacity of the shelfor were incorrectlyinstalled.

Your goal should beno skin contact withlaboratorychemicals.

4.1.f Moving ChemicalsA chemical sitting on a shelf in its original container presents less hazard than whenthat same container is moved. Whenever a chemical is moved there is a risk of thecontainer breaking resulting in an uncontrolled release. Most of the campus calls foremergency responders are the result of chemicals spilled while moving them withina building. Whether you are transporting chemicals across your lab or across thecampus, take precautions.

Use secondary containment. No matter how careful you are, containers can dropand bottles can break. An unprotected chemical container breaking in an elevatorcould be disastrous. Use a tray or a bucket to hold your chemicals in transit andcontain any possible accident. Good secondary containment can mean the differencebetween a small inconvenience and a major building evacuation. Check a laboratorysafety catalogue to find other secondary containment equipment to suit your needs.

Extra precautions for vehicles. The transportation of chemicals in vehicles onpublic roads presents additional safety and legal problems. A container of flamma-ble solvent or toxic material ruptured in a road accident drastically increases the riskto your health and makes rescue difficult. Chemicals should never be transported inthe passenger compartment of a vehicle. The state's Department of Transportation(DOT) and the Department of Natural Resources (DNR) regulate the transportationof hazardous materials on public roads. Depending on the type and quantity ofmaterial transported, the person driving may be required by law to have a special(e.g., commercial) driver's license, carry proper shipping papers and use specifiedpackaging. If you must transport hazardous chemicals on public roads, call Safetyfirst. We can give you guidance on how to do it safely. In some cases we may trans-port your materials for you.

Shipping Hazardous Materials. Many different government agencies regulatehazardous materials. If a hazardous substance is to be sent or transported off campus(e.g., FedEx, etc.), Department of Transportation (DOT) rules and regulations apply.The International Air Transport Association (IATA) sets rules for air transport ofhazardous materials. You must attend a training class and be certified to ship (i.e.,give to a commercial carrier like FedEx) any hazardous material. The SafetyDepartment offers training classes which will certify you for shipping chemicals orbiological materials. Call for additional information and a schedule of the training.

4.2 Chemical Storage and ManagementProper chemical storage is as important to safety as proper chemical handling. Don'tjust store your chemicals; your stocks and inventory of laboratory chemicals need tobe actively managed. From the time laboratory chemicals are received in your lab tothe time of disposal, inventory them, separate incompatibles, store them safely andregularly review their status.

4.2.a Chemical InventoryYour lab is in many ways like your home. Most people shop at the grocery storeusing a shopping list of food stuffs and consumables to buy. This shopping list isfilled out by taking inventory of what you have on hand and what your plans are forthe immediate future. In generating this list you consider things like, "When doesthe milk expire?" "If I buy 4 of these, how long will they last?"



Worker receivedburns to face andchest while carryingunsealed phenol-chloroform centri-fuge tubes on aStyrofoam centri-fuge tube shippingcontainer. TheStyrofoam brokeand the chemicalssplashed onto faceand chest. Evenimmediate delugedid not preventsecond degreeburns.

Secondary contain-ment means usinga second outercontainer to captureany possible leaksor spills.

Never transportchemicals in avehicle's passengercompartment.

Review yourchemical inven-tory. Annuallycheck the integrityof containers andlabels. For perox-ide formers, checkexpiration dates.Add water to keeppicric acid moist.Dispose ofunwanted,degraded or discol-ored chemicals. Ifyou find chemicalsin good conditionthat you no longerneed, offer them toothers who mightwant them, or givethem to the SafetyDepartment whowill try to redistrib-ute them to otherlaboratories oncampus.

Prudent management of chemicals in your lab is facilitated by an inventory.Such an inventory saves money because, with a knowledge of how much of whatchemicals are on hand, it prevents the purchase of duplicates. If you are aware ofusage levels, materials can be routinely ordered. It helps you monitor chemicals thatdegrade with age (e.g., ethers, gas cylinders, etc.), and it helps you keep incompati-bles separate. An inventory allows for sharing (e.g., Safety has a surplus chemicalredistribution program, most items have been unused) expensive compounds andallows for workers to anticipate special storage requirements that certain chemicalsmay have. Additionally, a summary of chemical types may provide emergencyresponders with a measure of hazards to be encountered within that lab.

You can keep information on each chemical container using a box of file cards ora computer database. A computer database can be shared with other laboratories inthe department / school to advertise surplus stocks and prevent redundant purchasingfor the entire department. Keep track of your chemical purchases by savinginvoices. Monitor chemical use by keeping track of empty bottles. Keep track ofchemical disposal by retaining copies of surplus chemical and carboy forms.

For your safety, it is important that you know your chemicals and the hazardsthey pose to you. For each chemical you purchase, you should also receive aMaterial Safety Data Sheet (MSDS). If you don't receive an MSDS, call the supplieror the Safety Department for a copy. Additionally, the Safety Department web page(http://www.fpm.wisc.edu/safety) has links to MSDS sites. Keep a binder of yourMSDS pages for everyone in your lab to use. Upon receipt of new compounds,review the MSDS to determine the hazards of the chemical. Chapter 2 of this Guidecan help you interpret the MSDS and other hazard information. Make sure thecontainer is labeled with the chemical's hazard, so that others know too.

On receipt is also a good time to update the inventory. In a large lab, the mosteffective system is to have a computerized inventory with each record correspondingto a single container. Essential inventory information includes:

w the chemical name as printed on the container,w molecular formula (for further identification and to simplify searching),w Chemical Abstract Service (CAS) registry number (unambiguously identifies

chemicals regardless of different names),w lot numberw source, andw size of container.

Other useful information for the database is:

w hazard classification to guide in safe storage,handling and disposal,

w date of acquisition to insure unstable compounds are not stored beyond theiruseful life and to insure older chemicals are used first,

w storage location (if multiple locations exist).

4.2.b Chemical Storage PrinciplesStoring chemicals requires the integration of several basic health and safety consid-erations. The inventory system goes a long way to assure safe storage along thelines of physical safety and chemical compatibility. Follow a few other basicconsiderations to assure safety.



Read chemical labels and MSDSs for specific storage instructions.The supplier of your chemical has already investigated safety issues and listed theseon the MSDS and label.

Store your chemicals in a safe place.Store chemicals in a well-ventilated area; however, don't store chemicals in a fumehood. Keep your fume hood clear for work requiring a fume hood. Use a ventilatedcabinet to store volatile and odorous chemicals. Use sturdy shelving with amplespace for every container. Never store hazardous chemicals in a public area or corri-dor. Don't store liquids above solids. To avoid the risks of lifting and reaching,keep large and heavy items on lower shelves. Store glass containers so that they areunlikely to be broken. Keep containers off the floor, safe from an accidental kick.Use plastic trays for secondary containment to contain liquid spills. Think aboutways to keep your chemicals from spilling and contained if they do spill.

Avoid storing chemicals on shelves more than six feet above floor.Store all hazardous chemicals below eye level so you can easily read the label. Donot store liquids above eye level. Fire codes require that nothing be stored within 18inches of a fire sprinkler head on the ceiling.

Keep incompatible chemicals separate.Group chemicals according to their hazard category (i.e., acids, bases, flammables,etc.) to prevent chemical reactions and insure compatible storage. Information ondetermining compatible storage of chemicals can be found at Appendix F. Somegeneral guidelines include:

üSeparate acids from bases, store these chemicals near floor levelü Isolate perchloric acid from organic materials. Do not store perchloric acid on a

wooden shelf.üSeparate highly toxic chemicals and carcinogens from all other chemicals. Place a

warning label on this storage location and keep it locked.üSeparate acids from flammables.üDo not keep peroxide-forming chemicals longer than 12 months.üDo not allow picric acid to dry out.ü If flammables need to be chilled, store them in a laboratory safe refrigerator, not

in a standard refrigerator.üFlammables should be stored in a flammable storage cabinet.

Label all chemical containers.If you make solutions, synthesize products or transfer chemicals to anothercontainer, make sure all containers are labeled. Clearly label each chemicalcontainer in your laboratory with:

w the chemical namew the principal hazard (e.g., carcinogen, irritant, corrosive, etc.)w the date prepared, opened or receivedw initials of the person making the label

OSHA and EPA rules and regulations imply that if a container has an expiration dateon the label, then the chemical must be used or disposed by that date. If you aregoing to hold chemicals past their expiration date, the viability of the chemical



The bottom shelf ofan organic chemicalstorage cabinetcollapsed. Thecabinet wasconstructed of thinplywood with parti-cleboard shelvesattached to apressed paper-board backing, notappropriate forchemical storage.

compound should be evaluated and documented for containers past their "expiration"date (e.g., write on the container a reevaluated expiration date and initial it).

Unfortunately, there are some routine laboratory environmental conditions thatcontribute to deface or obliterate labels:w hydrogen chloride vapor from neighboring chemicals.w Iodine from chemicals that hydrolyze to iodide and that is oxidized in air.w light, fluorescent and sun light, acts on red print.w embrittlement of paper.w cellophane tape over paper, tape discolors easily.w glue oxidizes.w mold grows on labels of bottles in cold rooms that condense air humidity.

While all chemical containers must be labeled to prevent the hazards and disposalproblems associated with unknown chemicals, labeling of many small vials withcomplete chemical names can be a difficult and tedious task. To make this jobeasier use these tips.ü Label the entire group. If you have a rack with vials that hold various fractions

from a column, label the entire rack with a description of what is contained in theindividual vials.

ü Refer to your notebook. Give the containers numbers that are referenced in yourlaboratory notebook.

Inspect storage areas periodically. It is not unusual when the Safety Departmentcleans out a lab to find chemical containers that are more than 20 years old, somewith labels that are unreadable. Storage areas should be inspected at least annually.Remove unwanted or expired chemicals and update the inventory. Visually inspectstored chemicals to determine viability and safety. Chemicals showing any of theseindications should be evaluated for disposal:

w slightly cloudy liquids w pressure buildup in containersw darkening or change in color w evidence of reaction with waterw spotting on solids w corrosion / damage to containerw caking of anhydrous materials w missing / damaged / illegible labelsw existence of solids in liquids or liquids in solids

4.2.c Storage of Flammable and Combustible ChemicalsChemical labels, Material Safety Data Sheets, and Chapter 2 of this Guide will helpyou identify flammable and combustible chemicals. Improperly stored flammableand combustible chemicals can provide the fuel that can lead to a catastrophiclaboratory fire.

Suppose you came home one day and found two 5-gallon cans of gasoline andseveral jars of paint thinner in your kitchen. Your first concern would be to movethe containers somewhere outside the house, making sure that you didn't break any.Then open the doors to remove the smell. Why would you be less concerned withsuch quantities of highly flammable liquids in your lab?



Chemical labels,MSDSs, andChapter 2 of thisGuide will help youto identify flamma-ble and combustiblechemicals.

For small contain-ers, label the entiregroup or numbercontainers andrecord in anotebook.

Recently the news reported the deaths of three firemen fighting a fire in a hardwarestore. The firemen were near the area where the store kept its 5-gallon cans of woodpreservative (e.g., Thompson's Waterseal). The cans exploded in the heat of the fire.Is it any wonder than firemen audit labs and continually ask what hazardous chemi-cals are in the building? Use the following guidelines for storing flammablechemicals:

w Minimize the amount of flammable liquids in your lab. Buy only what you willuse in the immediate future, and buy the smallest size that you need. Excessflammable solvents risk a fire, a dangerous spill and, if you are exposed to them,your health. Unused surpluses cost the University thousands of dollars each yearfor disposal.

w If a building or departmental flammable solvent storage room with a fire suppres-sion system is available, store flammable materials there until you need to usethem and remove only the amount needed for a particular experiment or task.

w In the laboratory, store flammables in a UL-approved (or equivalent) flammablestorage cabinet. Unless a cabinet is marked as approved for storage of flammableliquids, flammable solvents may not be stored there. In general, do not storeflammable liquids in cabinets below fume hoods or sinks.

w Store flammables, combustibles and other fuels away from strong oxidizers, suchas perchloric and nitric acids. It is best to store flammable liquids in an approvedstorage cabinet dedicated solely for that purpose. See Chapter 2 and Appendix Fof this Guide for a list of oxidizers.

w Limit quantities of flammable liquids stored outside of safety cans and flammablestorage cabinets to less than ten gallons per one hundred square feet (i.e., per labsuite). If you include flammables stored in safety cans and flammable storagecabinets, limit the amount of flammable liquids to less than twenty gallons per



A worker using aheat gun to heatapproximately 0.5 Lof heptane in aPyrex beaker byhand over an openbench. A splash ofheptane came incontact with theelements of theheat gun, ignitingthe heptane. Hetossed the beakeraway from him. Hissleeve caught fire,the flaming beakerlanded on anotherwork surface wherethe fire spread tothe computercontaining histhesis.

The Tragedy of a Laboratory FireIt is usually a small event that starts a laboratory fire. A hot plate is left on

or an unattended water bath dries out and overheats. These events ignite afuel source, and there are plenty of fuel sources in laboratories: papers,bench top covers, plastic labware, wooden shelves and, perhaps most danger-ous of all, flammable solvents.

It is easy to start a lab fire. It is easy to destroy a laboratory. All it takes isa lapse of memory or judgment (we're always in a hurry) or a carelesscoworker. Even a careless colleague down the hall threatens your life andwork.

If the laboratory door is left open, every room on the floor will be affected.Even if the fire is quickly extinguished, soot will cover papers, lab manuals,computer disks and equipment in neighboring labs. It is a reason for theentire department to be concerned about preventing fires.

In one year, 3 lab fires on campus cost $108,526 in damage, but thankfullyno lives. And these costs only take into account the cleanup of the room andthe replacement of equipment. What is the value of lost research notes,destroyed samples and time spent organizing another working laboratory?

What can you do? Beware of heaters and other fire hazards. Minimize theamount of flammable solvents stored in your room, and store those you mustkeep in a flammable storage cabinet. And keep your doors closed as much aspossible.

It is best to storeflammable liquids inan approvedstorage cabinetdedicated solely forthat purpose.

Limit quantities offlammable liquidsstored outside ofsafety cans andflammable storagecabinets to lessthan 10 gallons perone hundredsquare feet.

one hundred square feet of lab space. Thus, the maximum quantity of flammableliquids in each lab suite / fire area depends upon the storage configuration:* Glass, metal or plastic 10 gallons (38 liters)* Safety cans 25 gallons (95 liters)* Flammable liquid storage cabinets 180 gallons (684 liters)

w On your benchtop, limit the storage of flammable liquids to those in immediateuse. Handle flammable chemicals in areas free from ignition sources.

w It is best to store bottles of (flammable) liquids in a tray or pan (secondarycontainment) to catch any spills.

w Use plastic trays when storing chemicals in freezers. This prevents the bottlesfrom becoming embedded in ice and frost that often forms in freezers. It alsocontains spills and drips.

w Always bond metal containers to metal receivers when transferring large volumesof flammable liquids or gases.

w Static electricity can ignite flammable gases or vapors. If static electricity is aproblem, minimize static electricity by spraying with an antistatic agent. Usenonconductive materials (floors, mats, etc.) and grounding straps on instrumentsand machines, especially when transferring flammable chemicals between metalcontainers. These reduce the risk of generating static sparks. The greatest hazardfrom static electricity is in the winter when the air is dry.

w Never heat flammable chemicals with an open flame, use a water bath, oil bath,heating mantle, hot air bath, etc.

w Use a fume hood when there is a possibility of dangerous vapors.w Cold rooms pose a unique set of problems. One big problem with anything stored

in a walk-in cold room is that outside (hallway or room) air brings in moisturewhich condenses on everything that is cold. This will lead to mold which thriveson paper and glue of labels and can make stored containers "unknowns."

Take precautions when storing flammable chemicals in a refrigerator. Refrigera-tor temperatures are almost always higher than the flash points of flammable liquids(see 2.1.a). Compressors and circuits are often located at the bottom of the refrigera-tor where vapors from small spills or leaks can accumulate. Electrical sparks from a



Including thoseflammables storedin safety cans &flammable storagecabinets, limit theamount of flamma-ble liquids to lessthan 20 gallons per100 ft2 of lab space.

On your benchtop,limit the storage offlammable liquids tothose in immediateuse.

HNone of the limits may be exceeded in the combined column (e.g., 240 gal = 180 gal + 30 gal + 30 gal)HThese amounts include waste solvents'Distillation apparatus that is vapor tight and totally enclosed is considered a closed form

30 gal20 gal15 gal10 galChemicals actively being used inclosed' form (not exposed toatmosphere)H

30 gal20 gal15 gal10 galChemicals actively being used inopen form (exposed to atmosphere)H

240 gal180 gal120 gal60 galIn a flammable storage cabinet

m 38 oC(100 oF)

< 38 oC(100 oF)

BoilingPoint:

m 23 oC (73 oF)< 38 oC (100 oF)

< 23 oC(73 oF)

< 23 oC(73 oF)

FlashPoint:

CombinedClass I-CClass I-BClass 1-A

Maximum AmountH Allowed to bepresent in a control area:

Guidelines for Storage and Use of Flammable Liquids in Labs (summary)

conventional refrigerator can then ignite the flammable vapors that build up inside.Unless a cold room is ventilated and has a fire suppression sprinkler system, do notstore flammable liquids there. Two kinds of refrigerators are approved for storage offlammables:

1. Flammable liquid storage refrigerators. These have no spark sources within therefrigerator cabinet. There are, however, spark sources outside the refrigeratorcabinet from switches, motors, relays, etc. These spark sources can igniteflammable vapors present outside of the refrigerator. A bottle of flammableliquid that drops and breaks near one of these refrigerators can easily be ignitedby the sparks.

2. Explosion-proof refrigerators. These refrigerators are considerably more expen-sive because they have all spark sources completely sealed inside and are safe forflammable atmospheres both within and outside of the refrigerator cabinet.

In certain cases a conventional refrigerator can be modified by the Physical PlantElectric Shop to become a flammable liquid storage refrigerator. Only those refrig-erators that do not have a frost-free feature can be modified in this way. A frost-freerefrigerator incorporates heated coils and fans that cannot be removed from thecabinet.

Conventional refrigerators in laboratories and cold rooms are not safe forflammable storage and must be labeled "NO FLAMMABLES". Labels are avail-able from the Safety Department.

4.3 Personal Protective EquipmentWill a splash of caustic cause a small mess or a blinding injury? Your use ofpersonal protective equipment (PPE) could determine the answer. The Universityhas supply contracts with vendors who sell PPE and other safety equipment.Request a catalog to see the latest available equipment.

4.3.a Eye ProtectionEye injuries are horrifying, but preventable events.Wisconsin law requires eye protection for all laboratoryworkers, so no one should work or be inside a laboratorywithout proper eye protection. The Safety Department canorder prescription safety glasses for you under the statecontract for safety glasses. Many departments on campuswill pay the cost of prescription safety glasses that arerequired for work. Ask your supervisor for details.

Safety glasses are the minimum requirement for laboratory eye protection. Wearchemical splash goggles or a face shield over your safety glasses if there is a dangerof splashed liquids or shattering glass. Never wear a face shield without safetyglasses. If you work with ultraviolet or laser light, wear protective lenses specificfor the light's wavelength.

Materials Distribution Services sells nonprescription glasses and many safetyequipment vendors have a large assortment of eye protection. Newer models arenearly fog proof, comfortable to wear, come in a great variety of sizesand styles and are quite fashionable. There is no excuse for not wearing eye protec-tion in a laboratory.



Household refrig-erators are not safefor storing flamma-ble liquids.

Don't step into a labwithout wearingsafety glasses!

Should you wear contact lenses in laboratories? Be aware of the hazards ofcontact lenses. They are difficult to remove if your eyes must be washed in anemergency. Also, they can trap contaminants against your eye and they restrict theflow of natural fluids that remove minor eye contaminants. A better alternative forlaboratory work is to order prescription safety glasses through the SafetyDepartment.

4.3.b Respiratory ProtectionThe Occupational Safety and Health Administration (OSHA) has strict requirementsfor respirator (e.g., full-face mask or N-95 filter mask) use. Even a simple paperfilter mask is subject to OSHA rules. These requirements include a medicalquestionaire and a respirator fit test for all users. The is necessary because wearing arespirator increases the work of breathing, which may cause health problems forsome people. We also need to know the identity and concentration of the aircontaminant to select the appropriate filter and ensure it will properly protect you.

To avoid these problems, it is best to prevent inhalation exposures by usingengineering controls, (e.g., increased room ventilation, fume hoods and gloveboxes)rather than respirators. If you must wear respirators, contact the OccupationalHealth Office at Safety. We will help you conform to OSHA regulations.

4.3.c ClothingWear clothing that protects your skin. Shoes should completely cover your feet;sandals are risky because they don't cover your feet. Wear long pants instead ofshorts or skirts. Use a lab coat for further protection. The coat sleeves keepsplashes, aerosols and dusts from touching your forearm and wrist. Have a plastic orrubber apron available for working with strong caustics, corrosives or compoundswith a "skin" designation.

4.3.d Hand ProtectionVinyl and latex gloves should be a common sight in all laboratories. They areinexpensive, comfortable and provide a nominal barrier to common hazards. Checkthem for holes and change them frequently. Be aware that vinyl and latex glovesoffer no protection from many corrosives and organic solvents.

Use the Glove Chemical Resistance chart to choose the appropriate glove for thechemical you are working with. Be aware that even the variety of gloves in thischart does not afford protection from all chemicals. For aggressive chlorinatedsolvents, such as chloroform and trichloroethylene, order a glove like Silvershield®

or polyvinyl alcohol gloves from a safety equipment vendor.Remember that all gloves can be permeated by chemicals to some degree. They

are not meant to provide protection from prolonged immersion in chemicals. Usetongs to retrieve items from the bottom of your acid bath, don't routinely reach intoany liquid with a gloved hand. Someday your hand could go in deeper than the cuffor the glove could fail.

For reusable gloves, don't forget to wash or at least rinse off the gloves after use.This will prolong their useful life and prevent the spread of chemical contaminationfrom the dirty gloves. If you didn't remove the gloves after handling some things,wash them as you would your hands before touching anything like faucet handles ortelephones.



Avoid the problemsof using respirators.Use a fume hood,glove box or otherisolation device toprevent exposure toairbornecontaminants.

For laboratorychemicals, yourgoal should be zeroskin contact.



Key: E=Excellent; G = Good; F = Fair; P = Poor; NR = Not Recommended; NT = Not TestedNitrile = Ansell Edmont SolVex® nitrile latex; Natural = Marigold Industrial® natural rubber/neoprene unlined latex;Yellow = Best Value Masters® lined natural latexThis glove selection does not protect against all chemicals. For aggressive chlorinated solvents such as chloroform andtrichloroethylene, order some Silvershield® or polyvinyl alcohol gloves from a safety equipment vendor.

GPEMethanolEEESodium Hydroxide, 50%GNTELactic AcidEGEPotassium Hydroxide, 50%PFEKeroseneEENTCalcium HydroxideGFEIsopropanolEPEAmmonium HydroxidePNTEIso Octane

Common BasesNTFEIsobutyl alcoholNTEEHydrogen Peroxide

NRNTNRSulfuric Acid, 95%NTNTEHydrazineGEESulfuric Acid, 10%PNREHexane

NTEEPhosphoric AcidGENTGlycerineNTNTEPerchloric AcidPNTEGasolineNRNTNRNitric Acid, 70%NTFNRFurfuralNREENitric Acid, 10%EFEFormalinNTGEHydrofluoric Acid, 48%FEEEthylene GlycolENTEHydrochloric Acid, 10%FPNTEthyl AcetatePGEHydrochloric Acid, ConEGEEthanolGFFFormic Acid, 90%PPNRDioxane

NTFFChromic Acid, 50%FFGDioctyl PhthalateEFGAcetic Acid, GlacialPPEDimethyl Sulfoxide

Common AcidsPFNRDimethyl FormamidePPEDiethyl Ether

NRNRGXylene (Xylol)PNTNTDiesel FuelNTNTETurpentinePNTFDiethylamineNTNTETriethanolamineFGGDibutyl PhthalateNTNTETricresyl PhosphateNTGECyclohexanolNRNRNRTrichloroethyleneGEECitric AcidNRNRF1,1,1-TrichloroethaneNRNRNRChloroformNRNRFToluene (Toluol)NRNTNRChlorobenzeneNTNRNRTetrahydrofuranPPFCellosolve AcetateNTNTEStoddard SolventNTPGCellosolveNTENTSodium HypochloriteNRNRGCarbon TetrachlorideGPEPropyl AlcoholNTNRGCarbon Disulfide

NRNRGPerchloroethyleneGNTNTCalcium HypochloriteGGEOxalic AcidNTFEButyl Alcohol

NTNTEOctyl AlcoholPPFButyl AcetatePFNRNitrobenzeneNRNRPBenzene

NTNRENaphthas, VM&PPNTNRBenzaldehydeNTNRNRMorpholineNTGFAqua RegiaFNTEMineral SpiritsNRFNRAniline

NTPPMethyl MethacrylateGFEAmyl AlcoholPPPMethyl Isobutyl KetoneNTPEAmyl AcetateFPNRMethyl Ethyl KetonePPFAcetonitrilePNTEMethylamineEPNRAcetone

NRNRNRMethylene ChloridePNTPAcetaldehydeYellowNaturalNitrileChemicalYellowNaturalNitrileChemical

Glove Chemical Resistance

Glove TypesMany vendors offer protective gloves under various descriptions and brand names.These gloves normally fall into one of several compositions:

w Latex: Natural rubber, or latex is inherently elastic and resilient. It resists acids,alkalis, salts, and ketones. Natural rubber latex is blended with or dipped in otherpolymers to achieve a combination of features including the abrasion resistance ofnitrile with the flexibility of latex. Latex gloves are suited for food processing,electronics assembly, and laboratory chemical handling.

w Neoprene: Neoprene is a synthetic rubber developed as an oil resistant substitutefor natural rubber. It is resistant to acids, caustics, alcohols, inks, refrigerants,ketones, oils, fats, grease fertilizers, cleaners, and detergents. Neoprene glovesare used in petrochemical, degreasing, and refining, chemical processing, metalfinishing, mechanical work, painting, bleaching, and commercial dishwashing.

w Nitrile: Nitrile, a synthetic rubber, also referred to as NBR or acrylonitrile-butadiene. Nitrile gloves have superior puncture and abrasion resistance inaddition to chemical protection, and they will not weaken or swell in aromatic orpetroleum solvents, caustics, or animal fats. They are suited for chemical andfood processing, stripping and degreasing, motor and engine manufacturing,machining operation using cutting oil and coolants, electronics, and acid etchingand chemical washing.

w Norfoil: This lightweight and flexible laminate material resists permeation by awide range of solvents, acids, and bases. SilverShield® gloves are often usedunder other gloves, with chemical protective suits or when working with abrasivematerial. They are useful for chemical and petrochemical laboratory work, spillcleanups, and HAZMAT control operations.

w Vinyl: Also known as polyvinyl chloride or PVC, vinyl is a plastic material thatresists acids and alcohols, but not petroleum solvents. More economical thannatural rubber latex gloves, vinyl gloves are used for a variety of industrial andfood processing applications, intricate assembly work, laboratory research, andpharmaceutical manufacturing.

Permeation / DegradationRemember, all gloves are permeable. Permeation is a process in which chemicalsseep through glove material. It is similar to what happens to an air-filled balloon,the air gradually passes through (i.e., permeates) the balloon material and escapes.Similarly, a chemical can pass through a protective film without going throughpinholes, pores, or other visible openings. The individual molecules of the chemicalenter the film and “squirm” through by passing between the molecules of the glovematerial which may appear unchanged. Permeation data are presented in two values:

w Breakthrough time (minutes) is the time lapse between first contact of the chemi-cal and glove and the time to detection inside the glove. These times representhow long a glove can be expected to provide effective permeation resistancewhen totally immersed in the test chemical.

w Permeation rates are the highest flow rates recorded for the permeating chemicalsthrough the glove samples during a six-hour test. These qualitative ratings arecomparisons of permeation rates to each other.

Degradation is a reduction in one or more physical properties of a glove material dueto contact with a chemical. Certain glove materials may become hard, stiff, or



brittle, or they may grow softer, weaker, and swell to several times their originalsize. If a chemical has a significant impact on the physical properties of a glovematerial, its permeation resistance is quickly impaired.

There is no such thing as the “ideal” chemically resistant glove. Some laminategloves offer protection against a wide range of hazardous chemicals, there may belimitations in dexterity, tactile sensitivity, ability to grip when wet, tear and punctureresistance. Disposable gloves offer a decreased range of protection against hazard-ous chemicals but offer greater dexterity and mobility.

Multiple gloves can be worn together. Wearing one pair of thinner, more dexter-ous gloves over a flexible laminate combines the advantage of both. When usingthis approach, be sure to use the smallest laminate size that will fit comfortably.This allows the greatest dexterity when worn under the outside glove.

Immersion or prolonged contact is not common for chemical work in the labora-tory and reusable gloves do not need to be replaced very often. If used, inspect thesetypes of gloves before each use, and replace whenever they become discolored orshow signs of damage. Before reusable gloves are removed, thoroughly rinse themoff and allow to air dry.

Disposable gloves provide a barrier protection when working with small amountsof laboratory chemicals. If a disposable glove becomes contaminated, removeimmediately and replace with a new glove. Never reuse disposable gloves.

4.4 Reducing your Exposure to ChemicalsThe most important tool to reduce exposure to a toxic chemical is a complete knowl-edge of the chemical's properties. First, read and understand the Material SafetyData Sheet that should come with every purchased chemical. Call the Safety Depart-ment to obtain a copy of any MSDS you need, or to consult an MSDS of a chemicalthat you are considering obtaining. Before working with any unfamiliar chemical,review Chapter 2 of this Guide, reference books, call the Safety Department fortoxicity information or talk to experienced users of the chemical. Once you have athorough knowledge of the chemical, use appropriate controls to ensure its safe use.

4.4.a Fume Hoods and Other Engineering ControlsThe best way to reduce or eliminate one's exposure to airborne substances is theproper use of well designed engineering controls. With engineering controls, safetyis designed into the process and there is less reliance upon the skill and vigilance ofthe worker. Examples of engineering controls include chemical fume hoods, gloveboxes and remote automation that keeps a worker away from a dangerous process.For laboratories on campus, fume hoods are the most important and common type ofengineering control.

A biosafety cabinet (see figure, below) is not a chemical fume hood. Theirmotors are not explosion-proof and a biosafety cabinet should never be used forworking with flammable chemicals. If you are working with volatile toxic chemi-cals, special care needs to be taken because some biosafety cabinets do not ejectexhausted air from the building; they merely filter the air to remove airbornemicrobes. Chemical vapors and gasses easily pass through the filter and aredispersed back into the room.

A fume hood is usually a permanent installation in the laboratory, and shouldhave a UW-Madison Safety Department fume hood evaluation sticker affixed, and



Never storeflammable liquids inyour biosafetycabinet.

dated within the past year. A biosafety cabinet is a stand-alone piece of equipmentin the laboratory, sometimes connected to the building's exhaust ventilation. TheUW Environmental Health Unit on campus services and can certify the performanceof the biosafety cabinet in your lab. They recommend an annual recertification ofthe cabinet's performance.

How a Fume Hood WorksA fume hood's purpose is to assist in the safe handling of hazardous materials,especially those that produce vapors, gases, or dusts. Fume hoods provide ventilationto carry away airborne contaminants, and exhaust them outside of the building. Thefume hood's sash will also provide shielding to protect the user, and containment forsmall fires and explosions.

A fume hood must have an adequate face velocity (measured at the workopening) to ensure the proper removal of toxic materials. The currently acceptablestandard is 100 feet per minute (fpm) with a vertical sash opening of at least 18inches. The Safety Department annually checks the air flow of each of the approxi-mately 1700 chemical fume hoods on campus and mark the sash position at whichthe 100 feet per minute face velocity is attained at the fume hood's opening. Stickerson the hood show both air flow ratings and the date they were taken. The face veloc-ity must be at least 100 feet per minute to get a passing (i.e., green) sticker. Forwork with carcinogens or highly toxic chemicals, or for radioactive iodinations,greater velocities are required. Hoods which cannot meet the 100 fpm at the 18-inchopening, but can meet the 100 fpm rate at a sash height between 12 and 18 inches



w Airfoil - prevents dead space at front of hood, helpsprotect user from small spills.

w Light - located outside of hood or isexplosion proof.

w Bypass - provides constant face velocity independ-ent of sash position.

w Sash - provides mechanical protection for user.w Fan outside of building - ensures that ductwork inside building is under negativepressure.

w Baffles - provides air flow through face. Putadjustable baffles in center position.

w Fan and Stack - directs effluent upward athigh velocity from roof.

Fume Hood Features and Functions

The Safety Depart-ment annuallychecks the air flowof each of theapproximately 1700chemical fumehoods on campus.

may be approved for temporary, restricted use. Hoods which do not pass are unsafeand should not be used.

Although Safety checks and marks each fume hood, how can you be sure yourfume hood is functioning properly? A simple way to monitor your hood's operationis to do a tissue test to be sure air is moving into the hood. Tape a strip of tissue tothe edge of the sash. The tissue fluttering into the hood indicates air flow. This isonly a qualitative test for hood function (i.e., air moving or not moving into thehood). To properly evaluate a hood's performance, only a calibrated velometer willgive you an accurate quantitative measurement of face velocity.

Remember, air flow rate / velocity usually decreases as you raise the sash. Somebasic steps to insure an optimum airflow rate include:

ü Keep the sash as low as possible.ü Maintain air flow pathways front to back.ü Keep work more than 15 cm behind sash opening.ü Keep heaters more than 30 cm behind sash opening.



Other types of HoodsSash principles

If your ventilationsystem is modifiedin any way, callSafety to re-testyour hood.

If you doubt the efficiency of your hood, call the Safety Department, and ask for ageneral safety specialist.

Use of hot, concentrated perchloric acid requires a specially constructed hoodwith washdown facilities to avoid the build up of explosive metal perchlorates indownstream metal ductwork.

Guidelines for Maximizing Hood EfficiencyMany factors can compromise the efficiency of a hood operation. Most of these areavoidable; thus, it is important to be aware of all behavior that can, in some way,modify the hood and its capabilities. The following should always be consideredwhen using a hood:w Keep fume hood exhaust fans on at all times.w If possible, position the fume hood sash so that work is performed by extending

the arms under or around the sash, placing the head in front of the sash, andkeeping the glass between the worker and the chemical source. The worker viewsthe procedure through the glass, which will act as a primary barrier if a spill,splash, or explosion should occur.

w Avoid opening and closing the fume hood sash rapidly, and avoid swift arm andbody movements in front of or inside the hood. These actions may increaseturbulence and reduce the effectiveness of fume hood containment.

w Place chemical sources and apparatus at least 6 inches behind the face of thehood. In some laboratories, a colored strip is painted on, or tape applied to, thehood work surface 6 inches back from the face to serve as a reminder. Quantita-tive fume hood containment tests reveal that the concentration of contaminant inthe breathing zone can be 300-times higher from a source located at the front ofthe hood face than from a source placed at least 6 inches back. This concentrationdeclines further as the source is moved farther toward the back of the hood.

w Place equipment as far to the back of the hood as practical without blocking thebottom baffle.

w Separate and elevate each instrument by using blocks or racks so that air can floweasily around all apparatus.

w Do not use large pieces of equipment in a hood, because they tend to cause deadspaces in the airflow and reduce the efficiency of the hood.

w If a large piece of equipment emits fumes or heat outside a fume hood, then havea special-purpose hood designed and installed to ventilate that particular device.This method of ventilation is much more efficient than placing the equipment in afume hood, and it will consume much less air.

w Do not modify fume hoods in any way that adversely affects the hood perform-ance. This includes adding, removing, or changing any of the fume hood compo-nents, such as baffles, sashes, airfoils, liners, and exhaust connections.

Types of Fume HoodsFume hoods provide primary confinement in a chemical lab. They exhaust toxic,flammable, noxious, or hazardous fumes and vapors by capturing, diluting andremoving these materials. Fume hoods also provide physical protection against fire,spills and explosion. As noted, fume hoods provide the best protection when thefume hood sash is in the lowest practical position.

There are several types of fume hoods. Besides the standard fume hooddescribed above, there are also bypass and auxiliary air fume hoods.



üBe sure air ismoving intohood.üMaintain airflow

pathways.üKeep work > 6

inches behindsash.üKeep heaters >

12 inches behindsash.üClose sash when

not in use.üUse protective

gear as needed.üHave spill control

materials.üObey safety

labels and

w Bypass fume hoods, also called balanced air or constant volume fume hoods,allow a constant exhaust volume that helps keep the room ventilation systembalanced. Thus, as the sash is lowered, the same amount of air continues to enterthe hood through other openings. This technique also eliminates the problem ofhigh face velocity as the sash is lowered.

w Auxiliary air fume hoods, also called supplied air hoods, use a an outside airsupply for 50 - 70% of the hood's exhaust requirement. This type of hood isdesigned to reduce utility costs and conserve energy, but the face velocity of anauxiliary fume hood may vary with sash height.

Additionally, special purpose fume hoods are necessary when working with certainchemicals or chemical operations. These include:w Perchloric acid fume hoods have a water spray system to wash down the entire

length of the exhaust duct, the baffle and the wall. The water spray is usedperiodically or after each use to remove any perchloric acid or organic materialthat may have accumulated.

w Walk-in hoods have single vertical sashes or double vertical sashes and anopening that extends to the floor. These hoods are typically used to accommo-date large pieces of equipment.

w Canopy hoods capture upward moving contaminants and are good for heat-producing operations. In many ways these are like your stove-top exhaust fan.With this system, workers may be exposed to contaminants if they work underthe hood.

Alternatives to Chemical Fume HoodsChemical fume hoods are expensive to install and operate. Installation costsrange from $15,000-30,000, and operating costs (energy use) average $2,500 peryear to exhaust large amounts of heated or air-conditioned indoor air. Theexpense of a fume hood is well worth it to reduce your exposure when workingwith laboratory chemicals. But these costs are not justified when a fume hood isused to store chemicals or to vent laboratory equipment. There are alternativesfor storage and venting:

w Storing especially odorous or toxic chemicals in a ventilated storage cabinet cancontrol the odors and/or toxic vapors effectively, yet exhaust much smallervolumes of air resulting in a lower energy cost.

w Connect your instrument or apparatus directly to a dedicated exhaust duct that canhandle the effluent from the unit effectively while drawing less air.



Auxiliary Air Fume HoodBypass Fume Hood

A 36" wide hoodwith 100 fpm flow,moves air at 7.5ft3/sec or 0.21m3/sec. A l30' x 15'x 9' lab would get 7air changes perhour from that onehood. If the outsideis 25 1C differentthan the inside, thathood would wasteheat energy at arate of about 8.1kW.

w Flexible benchtop exhausters can be positioned very close to the source of anairborne contaminant and control it without exhausting the large volumes of airthat a fume hood requires.

The Safety Department strongly discourages the installation and use ofductless fume hoods. They allegedly control contaminants by passing themthrough a filter that "removes all contamination" before releasing the air backinto the room. But filters only work for specific substances and are never 100%efficient. Without continuous monitoring and testing of air contaminants, it isnearly impossible to tell when a filter fails or needs replacing.

4.4.b Work Practice and Administrative ControlsIn addition to engineering controls, exposure to hazardous substances can becontrolled by work practice and administrative controls.

Work practice controls manage the way workers use hazardous substances toreduce exposure. Examples include:

w Make sure containers of volatile chemicals have secure closures and are tightlyclosed when they are not actually being dispensed.

w Use smaller portions of hazardous chemicals by scaling down experiments.w Any other means that you can think of to reduce the generation of airborne

contaminants.w In essence, work smarter, be aware of the dangers of chemical exposure and

constantly search for ways to reduce them.

Administrative controls involve standard operating procedures and work rulesdesigned to minimize exposure to hazardous substances. Examples of administrativecontrols include:

w Limitations on the amount of time workers are allowed to use a substance.w A requirement that any new uses of a particularly hazardous substance be

reviewed by the supervisor or principal investigator prior to its use.w Write and follow a chemical hygiene plan. A chemical hygiene plan contains

standard laboratory operating procedures that are designed to limit exposure tohazardous laboratory chemicals (See Appendices B and C).

To be effective, administrative controls require a substantial commitment andfollow-up on the part of supervisors or the principal investigator.

4.5 Chemicals Requiring Special PrecautionsThis section deals with controlling specific chemical hazards common in manyresearch laboratories. For information on the types chemical hazards, see Chapter 2of this Guide. Particularly hazardous substances (see Chapter 3.1.b) requirespecial approval and controls that are described in Appendix D.

4.5.a Toxic Chemicals (Particularly Hazardous Substances)The OSHA laboratory standard (see Appendix B) requires that special precautions betaken when working with particularly hazardous substances such as chemicals withhigh acute toxicity, select carcinogens and reproductive toxins. Appendix Dcontains a more complete discussion of Particularly Hazardous Substances to includethe specific substances, the approval procedures, and the safety related items which



Keep your contain-ers closed whenthey are not beingused. The actualseal of thecontainer si theneck rim fittingevenly onto theliner of the cap.Tape around theoutside of the capdoesn't do the job.

should be considered to insure these substances will be used safely. If you use aparticularly hazardous substance, be sure to specifically address its storage, use,disposal and possible spillage in your laboratory's Chemical Hygiene Plan (seeAppendix C).

General PrecautionsConsult material safety data sheets and current references that list the toxic proper-ties of the substances that you work with. Employ all specified safety measures inaddition to the following general precautions:

Establish a Designated Area. Use and store these chemicals only in designated,restricted access areas. Post these areas with the appropriate warning signs. SeeAppendix D for a sample warning sign. Avoid working alone with these materials.

Chemical Storage and Management. Store breakable containers in chemicalresistant trays. Work over trays or plastic-backed absorbent paper. Keep an accuraterecord of your inventory of these chemicals. This inventory may include: amount ofchemicals stored, amount used, and the dates and names of people who work withthem. Store all hazardous waste in closed, labeled, impervious containers.

Use a Containment Device. Work in a fume hood when using volatilechemicals. Use a glove box if a procedure is likely to generate airborne dusts,aerosols or vapors. Consider setting up the glove box inside a fume hood to add amargin of safety should the glove box leak.

Personal Protective Equipment. Wear a double pair of impervious gloves andchange them frequently. Do not touch door knobs, telephones or computerkeyboards with contaminated gloves. When leaving the area, thoroughly wash yourhands, forearms and any other skin that may have been contaminated. Wear a longsleeved fully buttoned lab coat. If clothing becomes contaminated, decontaminate ordispose of it before leaving the area.

Waste Removal. See Chapter 7 and Appendix A of this Guide for detailedinstructions related to waste. Remember, if you are dealing with a listed hazardouswaste, dilution may only give you a larger volume of hazardous waste.

Decontamination Procedures. Decontamination is never as easy as it seems. Ifsurfaces become contaminated, you will need to consider such factors as surfacetype, spilled material, amount spilled, etc. Consider if the surface receiving the spillcan absorb the spilled material and if it can also absorb the solvent being used totake up the spill. If recovery of the material for use is not important, clean up is lesscomplex. It is usually best to remove as much contaminant as possible before usinga wet decontamination procedure or vacuuming dusts with a catch bag backed with aHEPA filter. Generally one should never dry-sweep powders, cover them with anabsorber first. Another possible method is to push tow dust pans or similar itemstogether to corral a powder onto one surface. Decontaminate any equipment orglassware removed from the area. See Procedure Labware 1 in Chapter 7 for decon-tamination and reuse of chemically contaminated labware.

Emergency Planning and Response. Be prepared for accidents or spills byreading Chapter 5. If a major spill occurs outside of a fume hood, evacuate the areaand call the Safety Department for assistance.

Prevent Exposure When Weighing Toxic ChemicalsThe act of weighing toxic chemicals for laboratory work often presents a risk ofexposure. For powders, static electricity can interfere with weighing. Air currentscan make powders airborne, which interferes with weighing and will risk your



Avoid spreadingcontaminationthroughout yourlaboratory.

Researcher usingnitrogen dioxide ina canopy hood withequipment attachedto a cold trap. Afterthe experiment wasfinished, researcherremoved cold trapfrom liquid nitrogento clean it in a fumehood. Whilemoving the trap, thefrozen NO2

sublimed and wasinhaled. Two dayslater the researcherexperiencedburning and tighten-ing in his chest.Cause: inadequateventilation.

exposure via inhalation. You may have to try several of the following PPE andsafety practices to find the combination that works for you. Your options tominimize exposure when weighing toxic chemicals include:

Minimize the frequency of weighing, and the amount of chemical used.Weigh out only what you need, consider larger batches to minimize the weighingfrequency and prepare the work area so it can be done efficiently.

Gloves. Always use gloves and wash your hands afterwards. If airborne powdersare a risk, wear a long sleeve lab coat to protect your arms.

Fume hood. A fume hood provides the most exposure protection. Use a fumehood to weigh all volatile toxic liquids. However, the air currents flowing throughthe fume hood may make it unsuitable for weighing powders. Alternatively, anenclosed, 0.1 mg accuracy pan balance, can be used in the hood where transfers fromthe stock container to the weighing container can be made. This will work for bothvolatile liquids and loftable powders, some of which are also susceptible to staticelectricity, if a pre-weighed, capped container is used. An unenclosed balance,which is used for larger amounts and has a 0.1 g accuracy, must be used outside ofthe hood to give a steady reading. The transfer to container or weighing paper ismade in the hood to protect the person from dust that flies or in the event of an"avalanche" which happens when a powder does not pour smoothly. Powder loss ispotentially greater in the air stream, but personnel exposure is minimized.

Biological safety cabinet. Although a biological safety cabinet (BSC) is notdesigned as chemical safety equipment, BSCs efficiently control and filter manyparticulates. Remember that a biological safety cabinet will not protect you fromexposure to volatile chemicals, and that flammable liquids should never be used in abiological safety cabinet.

Glove bag or box. Static electricity can be a problem. It is best to place these ina fume hood, although they can be used on a benchtop.

Air purifying respirator. An appropriate air purifying respirator can provideadditional protection, especially if you weigh toxic chemicals on a benchtop. Seesection 4.4.b for information on selection and use of air purifying respirators.

If you choose to openly weigh toxic chemicals on a benchtop, use an area that isaway from air currents, doors, windows and traffic and make sure you can detect anyairborne particulates (e.g., a light may help make them visible). Decontaminate thearea and balance after each weighing.

4.5.b Reproductive ToxinsUse extreme care with chemicals suspected of being reproductive toxins. Forpregnant women, even limited exposure can cause the death of the fertilized egg orfetus, retarded growth, malformation or deficits in postnatal function. Rememberthat pregnancy starts at conception, not after the first missed menstrual period. Aquestionable exposure should be discussed with your physician and stopped, ifadvisable, prior to conception. Other sources of information about reproductivetoxins are:

w The Reproductive/Occupational Health Consultant, Bureau of EnvironmentalHealth, Division of Health, State of Wisconsin Department of Health and SocialServices: 266-2074.

w Pregnancy Exposure Support Line of the Wisconsin Teratogen Project:1-800-362-3020.



4.5.c Chemical Carcinogens and MutagensWhen chemical carcinogens or mutagens (see Annex 4-1) are used, care is needed toprotect workers. The amount of a carcinogen or mutagen that will produce abiological response such as tumors and mutations depends upon many factors suchas the chemical structure of the agent, species and genetic constitution of theexposed individual, duration of exposure, route of administration, simultaneous orsequential exposure to other agents, etc. (cf., Chapter 2).

Because of these variables, it is difficult to predict the minimum amount of agentto implement safety precautions and it is prudent to handle all such chemicalscarefully, in properly designed and functioning laboratory. Adequate protectivemeasures must be taken to minimize the possibility of exposure, especially via skin,respiratory and digestive tracts, and eyes. Additionally, the more potent thecompound and the more compound that is handled, the greater the application ofprotective measures.

Antineoplastic DrugsMany drugs used as antineoplastic agents are in themselves carcinogens andmutagens (e.g., cyclophosphamide). Small quantities of these drugs are often usedin research laboratories as biological modulators. Aerosolization or direct skincontact are the primary routes of exposure during research or drug preparation andadministration. Handle antineoplastic drugs while wearing gloves and working in aClass II, Type A or B biological safety cabinet (BSC). If sterility is not a concern, afume hood can be used.

Reagent ChemicalsCertain reagents commonly used in biochemical and molecular biology laboratoriesare hazardous. One such chemical is ethidium bromide (see 4.5.i), a strong mutagenused in DNA sequencing and other molecular research. Handle hazardous reagentsin a fume hood while wearing gloves to prevent skin contact or breathing ofaerosols.

Laboratory ConcernsSafety begins with a quality laboratory facility and workers properly trained to usethe hazardous materials. As noted earlier, engineered facilities in conjunction withwork practices and administrative controls ensure safety.w Cover all work surfaces on which chemical carcinogens and mutagens are used

with stainless steel or plastic trays, absorbent plastic-backed paper, or otherimpervious materials. After use, decontaminate these surfaces with appropriatesolvent and dispose of waste products as described in Chapter 7.

w Perform procedures involving large amounts of chemical carcinogens ormutagens (e.g., syntheses) in a fume hood in metal pans which should be largeenough to contain all material in case of a spill.

w Use a properly functioning fume hood (see Section 4.4) for procedures thatinvolve volatile chemicals or that may produce aerosols from nonvolatile chemi-cals. Some aerosol-producing procedures include:

ü Inoculating and intubating animalsü Opening centrifuge containersü Mixing animal dietsü Transfer operationsü Blendingü Opening closed vessels



The fume hood must have a face velocity of at least 100 fpm when the windowsash is lowered to the designated working position for maximum air flow andshielding. While the Safety Department periodically measures face velocitiesand records the value on the hood, fume hood users should be alert to anydecrease or egress of air flow (e.g., use a piece of tissue to verify flow). Don'tuse a poorly functioning fume hood until repairs are made.

w If sterility is required, tissue cultures containing low-level amounts of carcino-genic chemicals may be manipulated in Class II, Type B biological safetycabinets that is vented to the building exhaust system. Class II, Type A biologicalsafety cabinets may be used only if adapted to discharge exhaust air to theoutdoors. For cultures containing larger amounts of carcinogens, Type B totalexhaust cabinets may be used.

w Never use clean benches or horizontal laminar flow cabinets for hazardouschemicals since air passing over the work area is directed at the user.

w Vent vapors or aerosols from analytical instruments (e.g., gas chromatographs)into fume hoods or Class II, Type B biological safety cabinets, or capture at theexhaust port with snorkels and duct into an exhaust system that vents outdoors.

w Exhaust and supply air openings should be kept unblocked and free of debris.w Hand-washing facilities should be available in the lab. Liquid soap is recom-

mended. Knee- or foot-operated apparatus is recommended for new laboratories.w Prominently marked emergency showers and emergency eyewash devices should

be readily available (see Chapter 5).

Operational PracticesThe best laboratory available is of little use if the workers do not implement goodwork practices. Procedures that help reduce risks include:w Only authorized personnel involved in research are permitted in the laboratory

(no casual visitors or children).w Keep access doors closed while work is in progress.w Inform maintenance and repair personnel of hazards and instruct them in precau-

tions if they are permitted entry.w If housekeeping staff are permitted entry, inform them of work areas they should

not clean and waste receptacles they should not empty.w Keep working quantities of chemical carcinogens to a minimum.w Label all chemical carcinogens and mutagens.w Designate specific work sites and storage areas by labeling.w Wear a fully-fastened, long sleeved laboratory coat and leave coats within the

laboratory before leaving.w Wear fresh gloves appropriate for the chemical carcinogen to be used. Wear

double gloving for especially hazardous compounds. Do not handle telephones,door knobs, etc., while wearing contaminated gloves. Remove gloves after use,discard in the appropriate waste container and wash hands.

w Wear eye protection when working in laboratories. Nonrefracting safety glassesor goggles are available from the UW Material Distribution Service. The SafetyDepartment provides prescription safety glasses at nominal costs for lab workers.

w Do not eat, drink, smoke, chew gum, or apply cosmetics in laboratory areas wherechemical carcinogens are used or stored. Keep hands away from mouth, nose,eyes, and face.

w Do not store food in laboratory refrigerators.



w Mouth pipetting is prohibited. Use mechanical pipetting devices.w Wash hands immediately after completing a procedure, after chemical exposure

or spill, and before leaving the laboratory.

DisposalChapter 7 discusses specific disposal chemical disposal methods and Chapter 8discusses animal use and disposal. Here we'll review some specific disposal consid-erations for carcinogens and mutagens.

Appropriate procedures for disposal of chemical carcinogens and mutagensdepend on the nature of the agent and the manner and quantity in which it has beenused. Properly identified wastes collected by the Safety Department will bedisposed of following all EPA and State DNR rules and regulaitons.

Residues of chemical carcinogens and mutagens on glassware can be rinsed withsolvent into carboys supplied by Safety for disposal of organic solvents. In this waymulti-gram quantities of carcinogens and mutagens can be safely destroyed. olventsmay be ethanol, acetone, or dimethylsulfoxide (DMSO). The amount of water in thecarboys should be kept as low as possible. If appreciable amounts of halogenatedhydrocarbons are used, they must be disposed of in a halogen approved carboy, alsosupplied by the Safety Department. See http://www.fpm.wisc.edu/safety or call theSafety Department (2-8769) for information on disposal methods, waste analysisforms, and collection times. There are also some agent-specific degradation anddisposal methods:w Alkylating agents (see Annex 4-1) and nitrosamides are usually readily

destroyed by alkaline solutions. In a fume hood, dissolve the agent in a water-miscible solvent (ethanol, acetone or DMSO) and slowly add an excess of 5%sodium hydroxide or sodium thiosulfate. After 24 hours the mixture should beneutralized, if necessary, and washed down the drain with large amounts of water.One word of caution, the treatment of N-Methyl-N'-nitro-N-nitrosoguanidine(MNNG) and related nitrosamides with alkaline substances should be carried outwith extreme care because toxic, gaseous diazomethane is produced.

w Aromatic amines, polycyclic aromatic hydrocarbons, and nitro-samines canbe degraded by incineration temperatures. Thus they can be discarded in theappropriate waste solvent jug if they are in solution.

w Ethidium bromide can be disposed of by adding water (100 ml water to 100 mgethidium bromide or 100 ml of a 1 mg/ml solution) and 50 ml of 5% sodiumhypochlorite (e.g., Clorox-type bleach). After stirring the mixture for 4 hours orallowing the solution to remain overnight in a fume hood, flush down a sewerdrain with ten times the volume of water.

w Aflatoxin can be destroyed by oxidizing agents such as 5% Clorox followed afterseveral hours by an equal volume of 5% acetone.

Emergency ProceduresIn the event of an emergency (accident or spill), appropriate action, first aid, anddecontamination are highly dependent on the nature of the material. Therefore, it isimportant to base an emergency response plan on the chemicals most likely to beencountered. Post an emergency spill protocol in all laboratories which specifiesprocedures, identifies appropriate response personnel to notify, and lists sources ofmedical aid. Chapter 5 of Guide discusses emergency procedures.



4.5.d Chemicals that have a Specific OSHA StandardBecause of their hazards, OSHA has specific standards for exposure to

w 2-acetylaminofluorenew 4-dimethylaminoazobenzenew N-nitrosodimethylaminew vinyl chloridew Inorganic arsenic (& arsenate salts)w leadw benzenew 1,2-dibromo-3-chloropropanew acrylonitrilew ethylene oxidew formaldehydew cotton dustw cadmium

w asbestosw coal tar pitch volatilesw 13 carcinogens including

4-nitrobiphenylw alpha-naphthylaminew methyl chloromethyl etherw 3.3'-dichlorobenzidine (& salts)w bis-chloromethyl etherw beta-naphthylaminew benzidinew 4-aminodiphenylw ethyleneiminew beta-propiolactone

If you work with any of the above chemicals, you need to be aware of and complywith the specific OSHA standards governing their use. These standards are abovethose required by the OSHA laboratory standard and, in some cases, may requirespecial signs, medical surveillance and routine air monitoring of your workplace. Ifyou use these chemicals routinely, even for short periods of time, you must haveyour workplace evaluated by the Safety Department to assure that your workpractices and engineering controls are sufficient to keep your exposures below theOSHA specified limits. The most common of these in laboratories are formaldehyde(formalin), benzene and ethylene oxide.

Formaldehyde is a potent irritant, a skin sensitizer and a carcinogen. Thecurrent Permissible Exposure Limit (PEL) for formaldehyde is 0.75 ppm and theodor threshold for most people is 1 ppm. If you can routinely smell formaldehyde inyour work area you may be overexposed.

First, try to find a less toxic substitute material. If you must use formaldehyde,perform all operations in a fume hood. Wear splash proof goggles and neoprene,butyl rubber or polyvinyl gloves. Remember, 100% formalin is 37% w/vformaldehyde.

We have found that formaldehyde vapor is very difficult to control. Air monitor-ing shows that it is often necessary to add ventilation and other engineering controlsto protect laboratory workers from dangerous levels of formaldehyde exposure. CallSafety if you have any questions about the health risks of formaldehyde.

Benzene is a known human carcinogen, with an OSHA Permissible ExposureLimit of 1 ppm. Generally accepted safety guidance for carcinogen exposure is tolimit one's exposure to the lowest level feasible. Laboratory use of benzene hasdiminished markedly in recent years, as xylene or toluene have been found to besatisfactory and much less toxic substitutes in most cases.

Ethylene Oxide is also a known human carcinogen, with an OSHA PermissibleExposure Limit of 1 ppm. It is a widely used sterilizing gas used for items thatcannot withstand steam autoclaving. EtO sterilizers should be connected todedicated exhaust ventilation that purges the sterilizer chamber outdoors before theoperator can open the chamber. This is the type of engineering control necessary toreduce ethylene oxide exposure to an acceptable level.



If you can routinelysmell formaldehydein your work areayou may beoverexposed.

4.5.e Precautions for Storing Osmium TetroxideOsmium tetroxide and sometimes ruthenium tetroxide are used in electron micros-copy. Because they are strong oxidizers, acutely toxic, volatile and difficult tocontain, their storage merits special precautions. Poorly stored osmium tetroxide isan unusually difficult disposal problem.

Normally all of the osmium tetroxide in an ampoule is used at one time, but ifsurplus needs to be stored it should be contained in the smallest container possible.This minimizes the amount of vapor in the head space and the release of volatilehazardous material when the container is opened.

Storage of concentrated solutions (1-5%) is a problem for the electron micro-scope labs because usually only small amounts are used at a time. Seal sparecrystals or solutions in glass ampoules, using a flame to melt the glass. To avoid theproblems of recontaining surpluses, we recommend that you purchase tetroxidesolutions in small, resealable ampoules. If you are uncomfortable with flame sealingampoules, a screw cap or crimp collar and septum container is satisfactory forstorage. It is essential that the cap or septum liner not react with osmium tetroxide(use polyethylene or teflon) and that it firmly contacts the bottle or vial rim. Thisrequires that the liner be cushioned to provide a seal when the cap is in place.

Wrapping a ground glass stopper with Teflon tape may adequately containosmium tetroxide if done carefully. A Teflon or polyethylene stopper in precisionground glass may also work. Plastic snap-on caps on smooth rim volumetric flasksare suitable if they fit correctly. Polyethylene snap-top tubes work as well, but theycan splash upon forcing open the snap-top. Other attempts to contain osmiumtetroxide usually fail; even the best ground glass stoppers allow tetroxide to leak.Silicone grease or parafilm wrapping only postpones the eventual leaking.

4.5.f Corrosive ChemicalsCorrosives are materials that cause destruction on contact with living tissue. Precau-tions for corrosives focus mainly on preventing such contact. Strong, concentrated or100%, acids and bases are especially dangerous. Aqueous strong acids (pH < 2) orbases (pH > 12) at greater than 1 molar, especially hydrofluoric acid, and strongorganic acids (pKa < 5) and amines (pKa > 9) are corrosive to skin. Eye protectionthat forms a complete seal around the eyes (goggles) and rubber gloves must alwaysbe used when handling corrosive materials. A face shield over safety glasses, arubber apron and rubber boots may also be appropriate. An eyewash and safetyshower must be readily accessible in areas where corrosives are used and stored.

Acids and bases should be stored separately. Organic acids should be stored withflammable materials, separate from oxidizers and oxidizing acids.

When mixing acids with water, slowly add the acid into the water. Acids thatgenerate heat during dilution, such as sulfuric, should be mixed over an ice bath toquench the heat. Concentrated solutions of hydrochloric and nitric acids produceslight warming when being diluted. Concentrated sulfuric acid, 18 molar, whendiluted to 3.5 molar can produce an 80 1C rise in water temperature.

4.5.g Reactive Chemical PrecautionsThe hazards of reactive chemicals are specific to each chemical's properties. Beforeworking with reactive chemicals, understand their dangers. Read the label for



recommended precautions. Consult references for specific information on theirreactivity and compatibilities. Chapter 2 provides an overview of reactive chemicalsand their identification.

With all reactives, use as small a quantity as possible. When working withreactive chemicals, use a hood sash, a safety shield or a face shield. Segregate yourreactive chemicals and store them away from heat and sunlight.

Oxidizers: Oxidation reactions are a frequent cause of chemical accidents.When stored, segregate oxidizers from flammable and combustible materials,organic material and other reducers.

Pyrophoric Chemicals: Pyrophoric materials (e.g., boranes, n-butyllithium,white phosphorus) ignite spontaneously on contact with air. Avoid a flammable spillby storing breakable glass bottles inside a rubber or plastic bottle carrier. Use andstore all pyrophorics in an inert atmosphere (e.g., stored under nitrogen or argon).

Shock Sensitive / Self Reactive / Explosive / Endothermic Substances: Atomsof these substances are bonded to each other in an arrangement that has high poten-tial energy. They can then spontaneously release large amounts of energy whenstruck, vibrated, dropped or agitated. An explosion results when a large amount ofenergy is released very rapidly, a sensitive explosive is one that explodes on slightprovocation. Examples of "slight provocation" includes temperature increase, shockof impact, grinding, vibration, or a chemical initiation. Chemical initiators include:redox; high nitrogen / oxygen content (e.g., peroxide, halogen oxide, exceptionallynitrated compounds, etc.); copper, silver and gold acetylides.

Some chemicals become increasingly shock sensitive with age. Inspect yourstock of reactive chemicals regularly to see if they are degraded and should disposedof those compounds which appear degraded or of which you are suspicious.

Laboratory accidents may occur from the inadvertent formation of explosive orshock sensitive materials such as peroxides from oxygen exposure on some chemi-cals, perchlorates from perchloric acid procedures performed in fume hoods (see4.6.g) and azides from azide salt solutions acting on lead in drains.

Mercury manometers that are used to gauge ammonia or acetylene pressure havealso been involved in numerous lab accidents. Mercury nitride (a.k.a. fulminatingmercury) or mercurous acetylide can be formed by mercury oxide residue, that maybe found on the outside of the manometer tube, coming in contact with the gas whilegauging its pressure. Both can explode when the manometer tube is tapped bysomeone while trying to get a reading from a "sticky" gauge.

Silver oxide and ammonia solution can produce a "fulminating silver" which isvery sensitive to movement and energetic in decomposition to its elements. Thissubstance also forms from left over, unreacted Tollins Test reagent. If the "silvermirror" test for sugars is done, reduce any left over reagent by immediately reactingthe reagent with an excess of a sugar.

Nitro organics such as nitromethane, nitrophenols and picric acid, form salts witha strong base (e.g., sodium hydroxide). These solid salts are both thermal and shocksensitive, producing quick, energetic decomposition.

4.5.h Peroxide-Forming ChemicalsCertain chemicals can turn into potentially dangerous, shock sensitive, organicperoxides with prolonged storage and/or concentration. One step to reduce the riskis to simply avoid the prolonged storage of all peroxide-forming chemicals. Use the



Do not modifyliterature proce-dures, especiallythose involvinghighly reactivecompounds, withoutproper technicalreview by otherscientists.

Inspect your stockof reactive chemi-cals regularly to seeif they are degradedand should bedisposed of.

Researcher heatinga mixture of alumi-num chloride,sodium azide anddry tretrahydrofuranon a hot platethought to be set at65 oC in a fumehood. Whileresearcher wasaway from thehood, the mixturedetonated and a fireensued.Cause: sodiumazide can explo-sively decomposeat temperaturesabove 275 oC, asteam or oil bathwas not used topromote evenheating.

table on the following page as a guideline to determine safe storage limits for thesechemicals.

The chemicals listed in the Peroxide-Forming Chemicals table (next page) canform polyperoxide chains or cyclic oligoperoxides that are difficult to detect andeliminate. These peroxides can come out of solution and form crystals or a gel in thebottom of the container. They are extremely unstable and can violently decomposewith the smallest disturbance, sometimes even spontaneously. Do not store thesechemicals longer than suggested, unless tests show that they contain less than 80ppm of peroxides.

The chemicals listed as hazard due to peroxide initiation of auto- polymerizationcan undergo explosive polymerization initiated by dissolved oxygen. Do not storethese chemicals longer than suggested, unless tests show that they contain less than80 ppm of peroxides.

The chemicals listed as peroxide hazard on concentration can form hydroperox-ides and ketone peroxides. These peroxides are soluble and can be detected withperoxide test strips or a KI-starch test. It is common to distill these peroxidizablesolvents before use and this concentrates the dissolved peroxides and subjects themto heat and mechanical shock. To safely distill peroxidizable solvents:

w Eliminate the peroxides with a chemical reducing agent or pass the solventthrough activated alumina.

w Add mineral oil to the distillation pot. This has the combined effect of "cushion-ing" any bumping, maintaining dilution and serving as a viscous reaction modera-tor in case the peroxides begin to decompose.

w Carefully monitor the distillation process to ensure that it does not dry outcompletely, and then overheat.

Reducing Peroxides During DistillationAdd small pieces of sodium metal to the distillation vessel to reduce peroxides. Usebenzophenone as an indicator for the presence of sodium metal (benzophenone, inthe presence of sodium metal forms a radical with a deep-blue color). When the bluecolor disappears, add more sodium metal. Be wary of high peroxide levels and waryof cleaning a "spent" pot.

If you have any of the peroxidizable solvents listed in the table that are older thanthe recommended shelf life, test them for peroxides if you can do so safely or callthe Safety Department for help.

Testing Peroxide-forming AgentsThe most hazardous compounds are those that form peroxides on storage withoutbeing concentrated. These are materials that can accumulate hazardous levels ofperoxides simply on storage after exposure to air. Peroxide-forming compounds thatconsist of vinyl monomers can form peroxides that can then initiate explosivepolymerization of the monomers. When they are stored as a liquid, the peroxide-forming potential increases and certain monomers (especially butadiene,chloroprene, and tetrafluoroethylene) should be considered a peroxide hazard onstorage. Thus, prior to working with a listed compound test the peroxide level.

1. Carefully examine the container for date of receipt and overall condition.Containers that show signs of oxidation (e.g., rusty container or cap) or are storedlonger than the recommended shelf life should be handled with extreme caution.



Worker attemptedto use anhydrousethyl ether in arotary evaporatorextraction. The4-liter container ofether was nearlyempty. Whilepouring the etherinto the apparatusin a fume hood, henoticed the liquidwas oily and had astrange odor.Deciding not to useit, he poured theether back into thecan and wenthome. The nextmorning he noticeda white residueinside the evapora-tor and used ametal spatula toscrape the residuefrom a glass joint.There was adetonation thatshattered the glass-ware. He was cuton the hands, face,ear and scalp. Hewas wearing safetyglasses and glasswas embedded inthe lenses. Thesash of the hoodwas cracked andthe light in the hoodshattered.

Do not open or move any of these containers. Call the Safety Department forassistance.

2. If the material is within the recommended shelf life, determine its peroxidecontent. Quantitative test strips are available from numerous vendors. They areas easy to use as pH paper.

3. Materials with peroxide levels less than 80 ppm may be used as usual.4. Any compound containing peroxides in excess of 80 ppm should be treated and

disposed. Call a Safety Department chemist for specific treatment and disposalmethods.

As with any procedure described in this Guide, the Safety Department chemists willdemonstrate peroxide testing or inspect a suspect container in your laboratory.

StyrenetetrafluoroethyleneVinyl acetate

ChlorotrifluoroethyleneEthyl acrylateEthyl vinyl ether

AcroleinAcrylonitrileAcrylic acidChloroprene (2-chlorobutadiene)

Liquids and liquified compressed gases that can be initiated by oxygen to polymerize. The usualperoxide test may not show peroxides. But there are indicators of polymerization. When inspectingliquids, look for an increase in viscosity. When inspecting gases, look for residue after evaporation ofa sample. Both of these may suggest a polymer.

FuranIsopropanolMethylcyclohexaneMethyl isobutyl ketoneTetrahydrofuranTetrahydronaphthalene

DicyclopentadieneDiglymeDiethyl ether (ethyl ether)1,4-DioxaneEthylene glycol dimethyl

ether (monoglyme)

Acetal2-ButanolCellosolves (e.g., 2-ethoxyethanol)Cumene (isopropylbenzene)CyclohexeneDecalin (decahydronaphthalene)

Common solvents that can accumulate peroxide products that are a risk on heating and concentration.Test for peroxides yearly.

Sodium amideVinylidene chloride

Isopropyl etherPotassium amidePotassium metal

ButadieneDivinyl etherDivinyl acetylene

High risk peroxide forming materials. Oxidation products are easily formed that are sensitive andenergetic. Examine these quarterly. The usual peroxide test will only work for isopropyl ether.

Peroxide-Forming Chemicals

4.5.i Ethidium Bromide (EtBr)Ethidium bromide is a powerful mutagen widely used in biochemical researchlaboratories for visualizing DNA fragments. It binds to single-, double- and triple-stranded DNA. Its primary mobility hazard is as a dust dispersed in air while beingtransferred as a powder from one container to another or while making solutions.Airborne dispersion is also a concern when cleaning spilled powder. Fortunately,ethidium bromide is brilliantly self indicating in its intense color. Contact with skinwill show.

There is no information on its rate of transport through skin. Mutagenic concen-trations demonstrated effects on DNA separations in cell division in fruit flies at1200 ppm and DNA damage in HeLa cells in culture at 16 ppm solution application.The threshold of noticeable color is about 16 ppm. Thus, any contact that stains the



skin should be washed off with soap and water until the stain is removed. Seekimmediate medical attention for dust inhalation that is at the level where color isseen in sputum or on a nose swab.

Ethidium bromide stains will bind to absorbents and gels so that these two timeswon't become sources of the dye. The sanitary sewer limit for ethidium bromide is10 mg/L (see Chapter 7). Solutions disposed via sanitary sewer will rapidly dilutebelow hazard levels before treatment collection is reached. Non-permeable surfaceslike glazed ceramics, metal and glass can be washed free of the dye and trashed,sharps can be disposed or reused, whichever is appropriate (see Chapter 9).

4.5.j MercuryMercury metal is one of the noble metals. It is quite stable to oxidation and occursnaturally, in some cases, as the metal. The oxide is easily decomposed to mercuryvapor at ~ 500 1C. The metal is uniquely volatile for a metal; vapor pressure is 2.5 x10-3 mm Hg at ambient temperature. This is may seem to be a low pressure to becalling mercury "volatile," but the hazards of breathing and skin absorption at thatconcentration are real.

Mercury spilled on the floor in a room will not result in equilibrium vaporpressure in any reasonable amount of time. Undisturbed evaporation of a 10 cmdiameter pool of mercury is estimated at 0.1 µg/sec, which gives very low levels ofvapor in lab air. Noticeable mercury exposures will occur whenever a thermometerbulb is broken and trapped in a rug that is then walked on. Vapor can be plumed intothe air and will accumulate if the ventilation rate is very slow, as is the case in an airtight house in the winter. People who constantly work in the contaminated spacewill need a few months to accumulate a hazardous blood level. However, mercuryaccumulation can be drastically hastened if a vacuum cleaner sucked the mercuryfrom a rug or floor and blew warm air over it while it was in the collection bag.Vacuuming could thus throw the whole amount into the air during the time themachine is running. Because the person vacuuming would be breathing at anelevated level, they could rapidly intake a large quantity.

High surface tension and poor "wetting" ability of liquid mercury makes handlingdrops of the liquid generally safe. Transdermal diffusion will occur when the skin isstained with a dispersion of the metal. People can be poisoned if they rub the metalinto their skin and leave it on for a while.

Mercury compounds are a totally different case. A notable example is the case ofthe mobile, volatile and penetrating dimethyl mercury. A few drops that can rapidlyabsorb into the skin of the hands can lead to slow death over months. Skin whiten-ing cream that have used mercury chloride as an ingredient have slowly invaded theusers system and produced toxic symptoms. A few tenths of a gram of mercury inthe body as a burden, whether rapidly acquired or slowly, can be lethal.

4.6 Laboratory Equipment HazardsRemember the proverb, "familiarity breeds contempt?" Your laboratory has manypotential hazards. Working within this environment day-after-day safely oftencauses workers to forget the potentially hazardous nature of their environment.Never underestimate the hazards associated with a laboratory.

Instruments and equipment that operate at high voltage, high pressures, highspeeds or high temperatures, are potentially dangerous. Such equipment should be



Worker received apotentially fatalelectric shock whenhe touched a highvoltage electricalconnector on anelectrophoresisdevice. Theseconnectors were inthe form of stack-able banana plugand the maleconnector plug wasleft exposed with noinsulation.

inspected and maintained regularly and serviced to reduce the hazards from failure.Many of the pieces of equipment you use daily can pose a hazard. When using anyequipment remember:ü use the correct equipmentü know how to operate the equipmentü inspect the equipmentü use the equipment properly

Equipment purchased for use in your lab is designed to be safely operated. Use theequipment for its intended purpose only. Do not modify or adapt equipment withoutguidance from the equipment manufacturer. Do not defeat, remove, or overrideequipment safety devices.

Be aware of the hazards of each type of equipment. Before using a new piece ofequipment, or one you have not used before, review the manufacturers literature tobecome familiar with how to operate the item and appropriate safeguards. Alwaysinspect equipment before using and keep the area around instruments and equipmentclear of obstructing materials.

Another potential hazard from operating equipment is aerosol production. anaerosol refers to the physical state of liquid or solid particles suspended in air.Aerosols containing infectious agents and hazardous materials can pose a seriousrisk because:ü if inhaled, small aerosol particles can readily penetrate and remain deep in the

respiratory tract,ü aerosols may remain suspended in the air for long periods of time, andü aerosol particles can easily contaminate equipment, ventilation systems, and skin.

Types of operations which may lead to the production of aerosols include:

w grinder, mortar and pestlew vacuum-sealed ampoulew syringe and needlew vortex mixerw pipetw sonicatorw magnetic stirrerw shakerw blenderw centrifuge

To reduce or eliminate the hazards associated with aerosols:w Conduct procedures that may produce aerosols in a biological safety cabinet or a

chemical fume hood.w Keep tubes stoppered when vortexing or centrifuging.w Allow aerosols to settle for one to five minutes before opening a centrifuge,

blender or tube.w Place a cloth soaked with disinfectant over the work surface to kill any biohazard-

ous agents.w Slowly reconstitute or dilute the contents of an ampoule.w When combining liquids, discharge the secondary material down the side of the

container or as close to the surface of the primary liquid as possible.w Avoid splattering by allowing inoculating loops or needles to cool before touch-

ing biological specimens.w Use a mechanical pipetting device.

4.6.a CentrifugesCentrifuging presents the possibility of two serious hazards: mechanical failure andaerosols. While the most common hazard associated with centrifuges is a broken



Never attempt tooperate a machineor instrument untilyour supervisor hastrained you inproper operatingprocedures andsafe use.

Disconnect anyequipment that isunsafe or does notwork properly, andremove it fromservice. Notify yoursupervisor of theproblem.

tube, given the high speeds achieved and the necessity to maintain balance it isimportant to properly load the unit, operate it only a speeds recommended by themanufacturer, wait until it has completely stopped before removing all of the sampleand properly cleaning the unit. The following guidelines help reduce accidents:ü When loading the rotor, examine the tubes for signs of stress and discard any

tubes that are damaged.ü Inspect the inside of each tube cavity or bucket. Remove any glass or other

debris from the rubber cushion.ü Insure that the centrifuge has adequate shielding to guard against accidental

flyaways.ü Use a centrifuge only if it has an interlock that deactivates the rotor when the lid

is opened.ü Do not overfill a centrifuge tube to the point where the rim, cap, or cotton plug

becomes wet.ü Always keep the lid closed during operation and shut down. Do not open the lid

until the rotor is completely stopped.ü Do not brake the head rotation by hand.ü Do not use aluminum foil to cap a centrifuge tube, it can rupture or detach.ü When balancing the rotors, consider the tubes, buckets, adapters, inserts, and any

added solution.ü Stop the rotor and discontinue operation if you notice anything abnormal such as

noise or vibration.ü Rotor heads, buckets, adapters, tubes and plastic inserts must match.

Some low-speed and small portable centrifuges may not have aerosol-tight chambersand may allow aerosols to escape. In these instances, use a safety bucket to preventaerosols from escaping. High-speed centrifuges pose additional hazards due to thehigher stress and force applied to their rotors and tubes. For high-speed centrifuges:

w Filter the air exhausted from the vacuum linesw Keep a record of rotor usage to reduce the hazard of metal fatiguew Frequently inspect, clean, and dry rotors to prevent corrosion or other damage.

Proper care and inspection of centrifuge rotors is important. Periodic cleaning of therotor and chamber is necessary to keep the unit in proper working order. Clean anyspills immediately. Other tasks which can assure safe use of centrifuges include:

w Review the rotor SOP to insure it includes the manufacturer's safety instructions.w Visually inspect the rotor for mechanical or chemical damage prior to each use.

Inspect the underside of the rotor, the web area of the rotor and the outer rim.Insure the top and bottom pieces of the rotor are tightly connected.

w Certain chemicals (e.g., phenol) attack plastic rotors and some nucleic acidextraction kits can damage the rotor. Chemical damage often appears as discol-oration, crazing, granulation, peeling or similar deterioration of the rotor finish.

w Mechanical damage such as cracks, scratches, or gouges can often be seen ordetected as an increase in noise or vibration during a spin. Do not ignore exces-sive vibration that does not resolve after rebalancing and checking the fit of therotor cover. Do not use the rotor if any damage or change is evident.



A Beckman ultra-centrifuge using alarge aluminumrotor was spinningmilk samples. Therotor failed due toexcessive mechani-cal stress and thesubsequent explo-sion completelydestroyed thecentrifuge. Thecause of the failureis believed to be theuse of a model ofrotor that was notapproved byBeckman for use inthat ultracentrifuge.

w Always use the rotor cover. Use an aerosol seal for containment of pathogenicmaterials. If you see scoring around the circumference of the top of the plasticrotor cover, replace the rotor, since this may indicate rotor deformation

4.6.b Compressed Gas CylindersAs noted in Chapter 2, compressed gases in the lab present chemical and physicalhazards. If compressed gas cylinders are handled incorrectly, they can be lethal. Abroken cylinder valve can cause a cylinder to act like a rocket. Exposure to somegases, such as hydrogen sulfide from a cylinder leak, can be lethal.

Storage of Large (50 inch) Compressed Gas Cylindersw Buildings where large gas cylinders are used should have a designated area for

storing newly received cylinders and empty cylinders for return to MaterialsDistribution Services (MDS). Use chains or straps to keep cylinders secured to awall or a cylinder rack.

w Never accept a cylinder if the name of the contents is missing or illegible.w Don't rely on color codes for identification. Don't accept a cylinder if it only has

a color code or the color code does not match the printed name.w Transport cylinders on a cylinder cart with a safety chain. Never move a gas

cylinder unless the cylinder cap is in place. Do not move a cylinder by rolling iton its base. Do not lift cylinders by the cap.

w Always secure gas cylinders to a wall or a stable bench with clamps, straps orchains. Fasten cylinders individually, not ganged, in a well ventilated area. Evenempty cylinders must be secured. Bench clamps are available from MDS.

w Don't remove protective cylinder caps until the cylinder is secured. Replacecylinder caps before returning cylinders to their storage area.

w A maximum of 3 flammable, oxygen or health hazard gas cylinders should bestored per 500 square feet of area.

w Store cylinders of flammable and oxidizing agents at least 20 feet apart.w Keep only the cylinders that are necessary for current work in your lab.w Keep cylinders away from all sources of heat, sparks, flames and direct sunlight

to prevent pressure increases. Do not heat cylinders to raise internal pressure.w Do not store gas cylinders in hallways or public areas.

Use of Gas Cylindersw Only use regulators approved for the type of gas in the cylinder. Do not use

adapters to interchange regulators and never use improvised adapters.w Always wear impact resistant glasses or goggles when working with compressed

gasses.w Never refill gas cylinders.w Limit the amount of highly reactive, toxic, or flammable chemicals to the quantity

necessary for planned experiments, or that will be used within a few months.Avoid the use of these compounds whenever possible

w Before using, check all connections under pressure for leaks. Swab connectionswith a soap solution and look for bubbles.

w Don't leave regulators and valves on corrosive gas cylinders except when they arein frequent use. Work the valve stem of a corrosive gas cylinder often to keep itfrom freezing.

w Do not force valve stems; they can easily snap off.



Always secure gascylinders to a wallor a stable benchwith clamps, strapsor chains.

w Turn off both the main valve and regulator when not using the cylinder.w Do not use copper (> 65% copper) connectors or tubing with acetylene, the acety-

lene can form explosive compounds with silver, copper and mercury.w Clearly mark empty cylinders "empty." Secure empty cylinders in your building's

designated cylinder storage area until they are picked up by the vendor.

Rent and Demurrage for Large Gas CylindersFor larger gas cylinders, there is usually a charge for the time the cylinder is inservice. Often this charge is in the form of demurrage, that is, a rental charge thatstarts after an agreed upon period of time. These charges are quite small on amonthly basis, but can add up over time. For example, a scientist on campus used acylinder of nitrogen from 1986 to 1992. The price of the gas was $8.82 but he paidover $135 in cylinder rental. To minimize these rental charges:w Order only the quantities of the gas that you need.w Keep track of the location of each cylinder and the date you received it.w Use your cylinders on a first-in first-out basis.w If you have no plans to use a cylinder for several months, it may be worthwhile to

return a partially full cylinder rather than storing it.

Precautions for Flammable GasesFlammable gases pose a double hazard. Restrict the use of flammable gases (e.g.,hydrogen, acetylene, propane, butane, etc.) to no more than three 220-ft3 cylinders offlammable gases or oxygen per 500 ft2 of unsprinklered laboratory space and use nomore than 400 ft3 of hydrogen gas in areas below ground level. If possible, useflammable gases only in areas where a flammable gas detection system is installed.Do not flare or burn off residual gas from laboratory equipment, duct the gas to ascrubber and neutralize it, or call the Safety Department for proper disposalinstructions.

Precautions for Lecture BottlesLecture bottles are small gas containers that can become a serious disposal problemfor the University. In 1993 the University spent $170,000 for analysis and disposalof 85 discarded lecture bottles ($2,000 per lecture bottle). This high disposal costreflects the hazards of laboratory gases and the difficulty of disposing of lecturebottles. Exotic and toxic gases (e.g., arsine, phosgene and nitrogen dioxide) areoften supplied in lecture bottles. The most expensive to dispose of are lecture bottlesthat are old, have inoperable valves or have no markings to indicate the contents.Old lecture bottles can leak or spontaneously rupture. Here are some steps to take tominimize the hazards and cost of lecture bottle disposal.

Annually inspect your lecture bottles. Examine your lecture bottles for theintegrity of their markings, tare weight tags and for corrosion. Use a soap solution(see above) to check for leaks at the valves. If labels and valve tags do not agree orif there is any question as to the contents, call Safety. Dispose of all lecture bottlesthat you have no plans to use in the immediate future. Call Safety for disposalinstructions (see Chapter 7).

Store them safely. Lecture bottles should be stored in a separate ventilatedcabinet where the temperatures do not exceed normal room temperatures. Becauselecture bottles may contain gases that are liquified at pressures below the 150 atmos-pheric limit, they can be more susceptible to increased pressure with heating;



In 1993 the Univer-sity spent $170,000for analysis anddisposal of 85discarded lecturebottles.

especially if critical temperature is attainable near room temperatures, lay them ontheir sides with their valves pointed toward the ventilation port.

Do not store corrosives with lecture bottles. The corrosive vapors of chemicalssuch as hydrochloric acid or nitric acid can destroy markings and damage valves.

Track their use. Attach a clipboard to the cabinet or a tag to the cylinder torecord dates and the weight of bottles before and after use.

Buy what you need; use what you buy. Buy only the amount of gas necessaryfor your work and use it all. Consider the efficiency of sharing gases with other labs.

Return unwanted or surplus cylinders or lecture bottles to the vendor. Somevendors will take back surplus gas and empty lecture bottles. When buying gases,ask the vendor about providing return service. Consider the high cost of disposal ifthe vendor will not accept surplus gases for return. Safety has lists of the vendorswho accept returned lecture bottles. We can also help you with packaging, labelingand shipping cylinders to be returned.

Give Safety your unwanted lecture bottles. If you have lecture bottles that willnot be taken back by the vendor call Safety for removal. Safety will dispose oflecture bottles not returned to the vendor. Don't mark a bottle "empty" unless youknow that it is actually empty.

4.6.c Cryogenic LiquidsCryogenic liquids are hazardous because of the physical and chemical characteristicsof their super-cooled state. Cryogenic liquids may cause explosions, fires, asphyxia-tion, tissue destruction or embrittlement of structural materials. Follow these guide-lines for using cryogenic liquids:

w Avoid skin and eye contact with cryogenic liquids. Always wear eye protection,preferably a face shield. Don't use gloves that can be frozen to the skin.

w Keep cryogenic liquids away from all sources of ignition.w Store cryogenic liquids in a well-ventilated area to avoid buildup of flammable

gases or the displacement of air. Do not inhale cryogenic vapors.w Store cryogenic liquids in double-walled, insulated containers (e.g., Dewar

flasks). Handle Dewar flasks carefully. Tape them thoroughly to prevent therelease of a large number of tiny glass slivers in the event the flask shatters.

w Pre-cool receiving vessels to avoid thermal shock and splashing.w Select work materials wisely. Cryogenic liquids alter the physical characteristics

of some materials. Accidents have been reported where Pyrex tubes have failedcausing injury.

w Use tongs to place and remove items in cryogenic liquid. Rubber and plastic maybecome very brittle in extreme cold, handle these items carefully when removingthem from cryogenic liquids.

w Use extreme care in transporting cryogenic containers. Use a cart for largecryogenic containers.

4.6.d Glassware UseBroken glass is one of the most common causes of laboratory injuries. To reduce thechance of cuts or punctures, use common sense when working with glassware.Inspect glassware for chips and cracks before use. When you cut glass, use handprotection and fire-polish all cut surfaces. Follow these additional tips to reduce therisk of injury:



Buy only theamount of gasnecessary for yourwork and use it all.

A 250 ml glass flaskover- pressurizedand burst, spraying2 workers withglass shards. Asmall leak / pinholein the flask devel-oped while beingfrozen and liquidnitrogen enteredthe flask. When theflask warmed, thenitrogen vaporized.

Cuts from forcingglass tubing intostoppers or plastictubing are the mostcommon kind oflaboratory accident.

ü Never use laboratory glassware to serve food or drinks.ü Use care in handling and storing glassware to avoid damaging it.ü Discard or repair any chipped or cracked items.ü Leave at least 10% air space in containers with positive closures.ü When possible, substitute plastic or metal connectors for glass connectors.ü Thoroughly clean and decontaminate glassware after each use.ü When inserting glass tubing into rubber stoppers, corks, or tubing:w Use adequate hand protection (e.g., gloves or a towel)w Lubricate the tubingw Hold hands close together to minimize movement if the glass breaks.

ü Use thick-walled, round-bottomed glassware for vacuum operations. Flat-bottomed glassware is not as strong as round-bottomed glassware. Carefullyhandle vacuum-jacketed glassware to prevent implosions. Dewar flasks, vacuumdesiccators, and other evacuated equipment should be taped or shielded and forvacuum work, use only glassware designed for that purpose.

ü Large glass containers are highly susceptible to thermal shock. Heat / cool largeglass containers slowly. Use Pyrex or heat-treated glass for heating operations.

With proper precautions, work with glassware can be conducted safely.

w When handling cool flasks, grasp the neck with one hand and support the bottomwith the other hand.

w Lift cool beakers by grasping the sides just below the rim. For large beakers, usetwo hands, one on the side and one supporting the bottom.

w Never carry bottles by their necks.w Use a cart to transport large bottles of dense liquid.

Regardless of the precautions you take, glass may still break. Broken glass poses ahazard for puncture wounds and injection of hazardous chemicals.

ü Do not pick up broken glass with bare or unprotected hands. Use a brush anddustpan to clean up broken glass. Remove broken glass in sinks by suing tongsfor large pieces and cotton held by tongs for small pieces and slivers.

ü Glass contaminated with biological, chemical or radioactive material must bedecontaminated before disposal.

ü Follow the guidance in Chapter 9 for disposal of glass and other sharps.

Frozen Glass Stoppers. Ground glass stoppers frozen by contact with base solutionsmay be welded hopelessly. Those welded by fluoride solutions may have built-uppressure of silicon tetrafluoride. Be careful when removing frozen glass stoppers.First try soaking the stopper in hot water to expand the glass. If this doesn't work,try a special solution for freeing frozen joints:w 10 parts chloral hydrate,w 5 parts glycerin,w 5 parts water andw 3 parts concentrated HClPaint the solution on the frozen ground glass joint or immerse into the solution. Ifyou need to remove a stopper by tapping, wrap the stopper in a cloth or paper toweland wear gloves to protect your hands and prevent injury in case of breakage.



Do not use chromicacid to clean glass-ware, use astandard laboratorydetergent. Chromicacid is extremelycorrosive. (seeChapter 6)

4.6.e Electrical SafetyMost of the recent laboratory fires on campus were started by the careless handlingof electrical equipment. Laboratories have stills, water baths and other apparatus thatcan overheat or cause electrical shocks. Minimize electrical safety hazards with thefollowing:

w Before use, check all electrical apparatus for worn or defective insulation andloose or broken connections. Power cords should be checked closely and replacedif defective.

w Connect all ground wires to clean metal, avoid painted surfaces. Use three-pronggrounded plugs whenever possible.

w Keep electrical wires away from hot surfaces.w Don't allow water and other potentially destructive liquids to leak on electrical

wires, switches and outlets.w Avoid the use of extension cords. University policy only allows extension cords

in temporary situations. The cord must be grounded.w Never touch a switch, outlet or other electric power source with wet hands.w Don't use homemade or makeshift wiring, call an electrician for wiring.

Electrophoresis equipment is an example of a system which may be a major sourceof electrical hazard. This is because there is both high voltage and conductive fluidwhich presents a potentially lethal combination. Workers may be unaware of thehazards associated with electrophoresis. A standard electrophoresis operating at 100volts can deliver a lethal shock at 25 milliamps. Even a slight leak in the device tankcan result in a serious shock. Besides the above, use these safety precautions toprotect yourself from the hazards of electrophoresis and electrical shock.

ü Follow the equipment operating instructions.ü Use physical barriers to prevent inadvertent contact with the apparatus.ü Use electrical interlocks.ü Do not disable safety devices.ü Use warning signs to alert others of the potential electrical hazard.ü Frequently check the physical integrity of the electrophoresis equipment.ü Turn the power off before connecting the electrical leads, opening the lid or

reaching into the chamber.ü Use only insulated lead connectors and connect one lead at a time using one hand

only. Insure that your hands are dry when connecting the leads.ü Keep the apparatus away from water and water sources.

4.6.f Heating SystemsNext to glass failure, the most common source of laboratory injury is the impropermanipulation of heating apparatus, particularly gas burners. These systems areneeded to provide the heat needed to effect a reaction or a separation. These include:

w microwave ovensw ovensw heating mantlesw ashing systemsw hot air gunsw hot platesw furnacesw oil and air bathsw open flame burners

Some laboratory heating procedures involve an open flame. Common hazardsassociated with laboratory heating devices include electrical hazards, fire hazardsand hot surfaces. When temperatures of 100 1C (212 1F) or less are required, it is



Most of the recentlaboratory fires oncampus werestarted by thecareless handling ofelectricalequipment.

Acetone spilled outof a reaction vesselduring the additionof dry ice. Itseeped underneatha nearby electronicbalance andignited.

Never leave anopen flameunattended.

safer to use a steam-heated device than an electrically heated device because they donot present a shock or spark hazard and they can be left unattended with the assur-ance that their temperature will never exceed 100 1C (212 1F). Follow these guide-lines when using heating devices:

ü Before using any electrical heating device insure that the heating unit has anautomatic shutoff to protect against overheating and that all connecting compo-nents are in good working condition.

ü Heated chemicals can cause more damage and more quickly than would the samechemical at a lower temperature.

ü Heating baths should be equipped with timers to insure that they turn on and offat appropriate times.

ü Use a chemical fume hood when heating flammable or combustible solvents.Arrange the equipment so that escaping vapors do not contact heated or sparkingsurfaces.

ü Use non-asbestos thermal-heat resistant gloves to handle heated materials andequipment.

ü Do not leave oil baths unattended. Place your oil bath within a plastic or metaltray to contain any spills.

ü Perchloric acid digestions must be conducted in a perchloric fume hood.ü Minimize the use of open flames.ü It is a good idea to connect all exit ports from gas chromatographs (GC), atomic

absorption (AA) spectrometers and other analytical instruments to an exhaustventilation system to exhaust toxic contaminants from the laboratory. Generallyspeaking, emission from a GC is not significant, but an AA that uses a flameshould be installed with a chimney anyway.

4.6.g Pressurized SystemsProcesses requiring high pressures present a physical hazard should the equipmentfail. High pressure operations should only be conducted in equipment specificallydesigned for this use and only by persons trained to use the equipment. Do notconduct a reaction in, or apply heat to, a closed system apparatus unless the equip-ment is designed and tested to withstand pressure (e.g., such systems may bestamped with safe operating pressures or have a plate attached with the information).Pressure systems should also have an appropriate relief valve. Additionally, pressur-ized systems must be fully shielded and should not be conducted in an occupiedspace until safe operation has been assured. Until safe operation is assured, remoteoperation is mandated. Points to remember for pressurized work:

w Minimize risk and exposure.w Identify and assess all hazards and consequences. In particular, consider how

failures may take place and their consequences.w Use remote manipulations whenever possible. Similarly, conduct the procedure

with equipment at locations remote from personnel.w Minimize pressure, volume and temperature. The stored energy available for

release is proportional to the total volume and pressure.w Design conservatively. Don't assume that the apparatus will have an "inherent

safety factor," sometimes this supposed factor will not be present.



A rule-of-thumb toremember:

Reaction ratesdouble for each10 oC (18 oF)increase intemperature.

Keep pressuresand volumes tothe smallestamount neededfor the procedure.

w Use material with a predictably safe failure mode. Seek material which will failin a ductile manner. Do not use a brittle material in an occupied area unless it isproperly shielded or barricaded. Remember, a failure is possible if the material isnot correct for the application, if fabrication is haphazard, or the right process isnot employed.

w Demonstrate structural integrity by a proof test. Ensure that the components ofthe pressurized system will maintain structural integrity at the maximum allow-able working pressure.

w Operate within the original design parameters. Do not exceed maximum allow-able working pressure (MAWP). Do not change working fluids or serviceenvironments without considering the consequences of a failure.

w Provide backup protection. Suitable pressure relief valves should be installed toinsure that the pressure level will stay within safe limits if the equipmentmalfunctions of is improperly operated.

w Use quality hardware. When buying material, insist on information about themanufacturer's design, test and quality control.

w Use protective shields / enclosures. These are essential if quality hardware is notprocured and they provide insurance for the unpredictable failure.

w Use tie-downs to secure tubing, hoses, and piping. Remember, if a line failsunder pressure it can whip about unless it is restrained.

w Do not leave a pressurized system unattended.

4.6.h Vacuum SystemsVacuum systems may have similar hazards as high pressure work. Here, vacuumlines and other glassware at sub-ambient pressure may implode. flying glass is notthe only hazard. Dangers associated with possible toxic chemicals contained in thesystem as well as fire (e.g., of a solvent stored in the container). Precautions to helpinsure safety:

ü Insure that pumps have belt guards in place during operation.ü Insure that service cords and switches are free from defect.ü Always use a trap on vacuum lines to prevent liquids from being drawn into the

pump, house vacuum line, or water drain.ü Replace and properly dispose of vacuum pump oil that is contaminated with

condensate. Used pump oil must be disposed to UW Safety (see Chapter 7).ü Place a pan under pumps to catch oil drips.ü Do not operate pumps near containers of flammable chemicals.ü Do not place pumps in an enclosed, unventilated cabinet.

Glassware used in vacuum operations may pose a hazard if it breaks. To reduce theinjury from glass debris:

w Only heavy-walled round-bottomed glassware should be used for vacuum opera-tions. The only exception is glassware specifically designed for vacuum opera-tions (e.g., Erlenmeyer filtration flask).

w Wrap exposed glass with tape to prevent flying glass if an implosion occurs.w Carefully inspect vacuum glassware before and after each use. Discard any glass

that is chipped, scratched, broken, or otherwise stressed.



Pressurizedsystems shouldnot include glasscomponentsunless they arespeciallydesigned andintended for thatpurpose.

All vacuum equip-ment is subject topossible implo-sion, conduct allvacuum opera-tions behind atable shield or in afume hood.

Glass desiccators may develop a slight vacuum due to contents cooling. Whenpossible, use molded plastic desiccators with high tensile strength. For glass desic-cators, use a perforated metal desiccator guard.

Vacuum pumps often have a cold trap in place to prevent volatile compoundsfrom getting into hot pump oil and vaporizing into the atmosphere and to preventmoisture contamination in a vacuum line. Guidelines for using a cold trap include:

w Locate the cold trap between the system and vacuum pump.w Insure the cold trap is big enough and cold enough to condense vapors present in

the system.w Check frequently for blockages in the cold trap.w Use isopropanol/dry ice or ethanol/dry ice instead of acetone/dry ice to create a

cold trap. Isopropanol and ethanol are cheaper, less toxic, and less prone to foam.w Do not use dry ice or liquefied gas refrigerant bath as a closed system, these can

create uncontrolled and dangerously high pressures.

4.6.i Distillation of Organic SolventsPotential hazards from distillations arise from pressure buildup, flammable materials,and the use of heat to vaporize the chemicals involved. Care must be taken duringconstruction of the system to insure effective separation and to avoid leaks whichcould lead to fires or contamination. Take precautions with distillations andreactions, especially when they run overnight. Use these guidelines for safedistillations:

ü Prevent overheating by ensuring that all hoses and connections are securely tight-ened.

ü Always leave a phone number where you can be reached. Post it on the door ofyour lab so that emergency responders can contact you for information in case ofa fire or emergency. See the section on emergency posting in Chapter 5 of thisGuide.

ü Use boiling chips or stir bars to prevent bumping during distillations, refluxing,and similar procedures.

ü Be aware when distilling chemicals that certain types may autoxidize andaccumulate peroxides. Peroxides can explode when heated and concentratedduring a distillation. For more information on peroxides see Section 4.5.g.

ü Use only round-bottom flasks for vacuum distillations. Erlenmeyer flasks aremore likely to implode. Vacuum distillations or evaporations should always beshielded in case of implosion.

4.6.j Refrigerators / FreezersAs noted earlier (cf., 4.2.c), using a household refrigerator to store laboratory chemi-cals is hazardous for several reasons. Many flammable solvents are still volatile atrefrigerator temperatures. Refrigerator temperatures are typically higher than theflash point of most flammable liquids. Additionally, the storage compartment of ahousehold refrigerator contains numerous ignition sources including thermostats,light switches and heater strips. Furthermore, the compressor and electrical circuits,located at the bottom of the unit where chemical vapors are likely to accumulate, arenot sealed.

Laboratory-safe and explosion-proof refrigerators typically provide adequateprotection for chemical storage in the laboratory. For example, laboratory-safe



refrigerators are specifically designed for use with flammables since the sparkingcomponents are located on the exterior of the refrigerator. Explosion-proof refrig-erators are required in areas that may contain high levels of flammable vapors (e.g.,chemical storage rooms). These guidelines will help insure safety:w Never store flammable chemicals in a household refrigerator.w Do not store food or drink in a laboratory refrigerator / freezer.w Insure that all refrigerators are clearly labeled to indicate suitable usage.w Laboratory-safe and explosion-proof refrigerators should be identified by a

manufacturer label.w Refrigerators used to hold food should be labeled, "For Food Only."

4.6.k Autoclave SafetyAutoclaves are used in many areas to sterilize materials by high heat and pressure.The hot (132 oC [270 oF]), pressurized (30 psi) steam that autoclaves generate makethem serious burn hazards as well. Burns can result from physical contact with theautoclave structure and from contact with the steam leaving the unit. Explosivebreakage of glass vessels due to temperature stresses can produce mechanical injury,cuts and burns during opening and unloading the unit. Burns can also result fromcareless handling of vessels containing hot liquids. Additionally, because of theextreme conditions created inside steam autoclaves, they can easily malfunction ifnot carefully maintained. Because each autoclave make / model has unique charac-teristics, it is imperative that you read and thoroughly understand the manufacturersoperating procedures before you use an autoclave for the first time.

An autoclave uses different patterns of high heat, vacuum and pressure to sterilizematerial. The main types of runs are:ü liquids, for any type of water-based solutions,ü dry goods with vacuum, andü dry goods without vacuum

Autoclaves often have an additional drying cycle in which hot air is drawn throughthe chamber to dry materials after sterilization. Remember, controls for differentbrand of autoclave vary making it important to carefully follow the manufacturer'sinstructions about loading, load sizes, and cycle types and settings.

The liquids run is longer than the other two types, but uses lower temperatures tominimize evaporation of the liquids being sterilized. Insure seals on liquid contain-ers are loose so any expanding vapors produced during heating will not cause anexplosion. Use a tray with a solid bottom and walls to contain the bottles and catchspills. Never autoclave any flammable or volatile liquids.

The dry goods with vacuum run moves steam and heat into the deepest part oflarge bags or bundles of materials and produces the best conditions for killing persis-tent organisms. During this procedure, they chamber alternates between cycles ofhigh pressure, steam and vacuum. It is important that steam and pressure be able toreach the entire load, so carefully loosen bag closures once they are in the autoclave.

The dry goods without vacuum run simply pressurizes the chamber with steam forthe duration of the cycle and then returns to normal. This process is primarily usedfor items that have been cleaned but need to be sterilized. Materials should bepacked so that the heat and pressure can readily reach the whole load.

Autoclaves generate extreme heat and high pressures, users should understandand respect the hazards these create. To prevent a sudden release of high-pressure



Do not store foodor drink in labora-tory refrigeratorsunless labeled"For Food Only"

steam, firmly lock autoclave doors and gaskets in place before you run the autoclave.Most, but not all autoclaves, have safety interlocks that prevent operation if the doorisn't closed properly. Know if your autoclave has interlocks and take extra precau-tions if it is not equipped with interlocks. Some older autoclaves have little or noheat shielding around the outside. For these systems, attach Hot Surfaces, KeepAway warning signs to remind people of the hazard. Do not stack or store combus-tible materials (e.g., cardboard, plastic, volatile or flammable liquids, etc.) next to anautoclave. When operating an autoclave, follow these precautions:w Load the autoclave properly. Be sure to clean the drain strainer before loading.

Don't load plastic materials that are not compatible with the autoclave. Individualglassware pieces should be within a heat resistant plastic tray on a shelf or rack,never place them directly on the autoclave bottom or floor.

w Be sure the autoclave is OFF and the steam pressure is down before opening thedoor.

w Open the door slowly, keeping head, face and hands away from the opening.w Wait at least 30 seconds after opening the door before reaching or looking into the

autoclave. Before removing autoclaved items, wait 5 minutes for loads contain-ing only dry glassware and 10 minutes for autoclaved liquid loads.

w When removing items from the autoclave, wear heat-resistant, long-sleevedgloves and safety glasses or goggles treated with anti-fog solution. Removesolutions from the autoclave slowly and gently, some solutions can boil overwhen moved or when exposed to room temperature.

w Let glassware cool for at least 15 minutes before touching it with ungloved hands.Be alert for autoclaved liquid bottles still bubbling. Let liquid loads stand in anout-of-the-way location for a full hour before touching them with ungloved hands.

w Clean up any spills immediately.

4.7 Review Questions

1. To help ensure a safe laboratory, always make sure that your lab is:a. Neat, clean and orderly.b. Never stocked with more hazardous chemicals than you can possibly use within 1 year.c. Well equipped with a nearby eyewash station, safety shower, fire extinguisher and fire alarm so

that you are ready when an emergency occurs.d. Staffed by well-trained workers, who know about the hazardous properties of the chemicals they

work with, how to work with them safely, and what to do in an emergency.e. All of the above.

2. Horseplay and practical jokes in a laboratory is:a. Common practice.b. Very dangerous and strictly forbidden.c. Okay on April Fools' day since everybody else is doing it.d. Unprofessional and should never be allowed.e. b and d.



3. Before you open a container of any hazardous chemical you should:a. Find out if there is a less hazardous substitute for the chemical.b. Find out all that you can about the physical, chemical and toxicological properties.c. Have a plan for using the chemical, and know the proper method of disposal.d. Write the date you opened it on the label, so that someone else will be able to determine if the

shelf life of the chemical has expired.e. All of the above.

4. Today's properly attired lab worker is wearing:a. A Tyvek suit.b. Closed-toe shoes, safety glasses with side-shields, a lab coat, and gloves that have been selected on

the basis of what chemicals are in use.c. Natural fibers, such as cotton or wool.d. A three-piece suit and necktie. (clip-on, of course).e. A self-contained breathing apparatus and a fully encapsulating chemical resistant suit.

5. Flammable liquids should never be stored:a. In paper cups.b. On your lab bench top.c. In a conventional refrigerator.d. In your lab in large quantities (greater than ten gallons).e. All of the above.

6. Regarding water and concentrated acids, "Always:a. Add acid to water, like you oughta."b. Add water to acid, then watch it bubble over."c. Have a bucket of water available for emergencies."d. Dispose of acid by rinsing it down the drain with lots of water."

7. Corrosives are materials that cause tissue destruction on contact. Corrosives include:a. Acids with a pH [ 2. b. Bases with a pH m 12.c. Mineral oil. d. Saline solution. e. a and b.

8. Protective equipment required for using corrosives in a laboratory includes:a. A respirator.b. Steel-toed shoes.c. Goggles that form a seal completely around the eyes.d. Heavy-duty rubber gloves.e. c and d.

9. According to State fire codes, the maximum number of large four foot tall oxygen, flammable, orhealth hazard gas cylinders allowed per 500 square foot laboratory area is:a. Five. b. Twelve. c. Three.d. As many as you want, as long as they are securely chained.

10. Formaldehyde is:a. A potent irritant. b. A skin sensitizer. c. A carcinogen.d. Something you should only use under carefully controlled conditions, in a properly operating fume

hood.e. All of the above.



11. A chemical fume hood's purpose is:a. For storage of chemicals.b. A display case for fancy lab equipment.c. To assist in the safe handling of hazardous materials that represent an inhalation hazard.d. To contain small explosions that may occur in certain laboratory operations.e. c and d.

12. Responsibility for laboratory safety on the UW Campus lies with:a. Students and employees.b. Faculty and staff.c. The Safety Department.d. All of the above.

13. Employees and students are responsible for:a. Making sure that all fire extinguishers are inspected once a year.b. Making sure safety glasses are available for everyone in the laboratory.c. Reading Material Safety Data Sheets for every chemical they work with.d. Writing a Chemical Hygiene Plan for their laboratory.

14. When transporting hazardous chemicals on the highway:a. Keep them safely stored in the passenger compartment of your vehicle.b. Call the Safety Department first.c. Keep all containers hidden from view.d. Have another person accompany you.

15. When distilling organic solvents:a. Use an Erlenmeyer flask.b. Stay out of the room.c. Make sure every last drop is boiled off.d. Use boiling chips or stir bars to prevent bumping.

16. A refrigerator for storing flammable chemicals must be:a. Set at a very cold temperature.b. Frost free.c. Vented.d. Have no spark sources inside.e. All of the above.

17. Chemicals can be safely stored by:a. Putting them up high, out of the way.b. Keeping them on the floor.c. Putting them as close to a fire suppression sprinkler as possible.d. None of the above.

18. Flammable liquids can be safely stored:a. In a cabinet under a fume hood.b. In a flammable storage cabinet.c. In a functioning biosafety cabinet.d. All of the above.



19. Wisconsin law requires that:a. No contact lenses are worn in a chemical laboratory.b. At least one respirator is available in every laboratory.c. Eye protection is worn by everyone in a laboratory.d. All of the above.

20. A good way to tell if a fume hood is running is:a. Check the indicator light.b. Use the "tissue test."c. Look at the inspection labels.d. All of the above.

21. When working with particularly hazardous chemicals (Appendix D):a. Establish a designated area.b. Use a containment device.c. Wear a double pair of gloves.d. Have brush available to sweep up any spilled powders.e. All of the above.f. a, b and c only.

22. When working with autoclaves, both the high temperature and high temperature steam are a hazard.It is prudent to:a. Wait at least 30 seconds after opening the door before looking into the autoclave.b. Before removing autoclaved material, wait at least 5 minutes for dry glassware and 10 minutes for

liquids.c. Let glassware cool at least 15 minutes before touching it with ungloved hands.d. All of the above.

23. Laboratory refrigerators used to hold food should be labeled:a. "No flammables."b. "For Food Only."c. "Food O.K. in refrigerator compartment."d. "No Chemicals."

24. One of the most common causes of laboratory injury is:a. Inoculation with an infectious or carcinogenic agent.b. Explosion from flammable gas cylinders.c. Cuts from broken glass.d. All of the above.

25. Cryogenic liquids are hazardous because of their super-cooled state. Hazards include:a. Embrittlement of materials.b. Freezing of equipment to unprotected skin.c. Lung damage from inhalation of cryogenic vapors.d. All of the above.



26. Types of operations which may produce an aerosol include:a. centrifuge b. blender c. shaker d. sonicatore. pipet f. vortex mixer g. vacuum-sealed ampouleh. All of the above.

27. Centrifuging presents two types of hazards:a. aerosol production.b. sample implosion.c. mechanical failure.d. a and c.



Annex 4-1. Experimental Carcinogens and Mutagens

Although some of these examples are active per se, the majority require enzymatic activation. Storethese agents dry and away from light. Agents that should also be kept in a refrigerator are marked withan asterisk (*).

Alkylating agents (active per se)Ethylmethanesulfonate (EMS) (volatile liquid)Methylmethanesulfonate (MMS) (volatile liquid)Dimethylsulfate (volatile liquid)Diethylsulfate (volatile liquid)β-Propiolactone (volatile liquid)

Aromatic amines and derivatives2-Acetylaminofluorene (AAF)*N-Hydroxy-AAF*N-Acetoxy-AAF (active per se)2-Naphthylamine (β-Naphthylamine) (known human carcinogen)Benzidine (known human carcinogen)4-Aminobiphenyl (known human carcinogen)4-Nitrobiphenyl (known human carcinogen)4-Nitroquinoline-l-oxide

NitrosamidesThese are highly potent agents. Homologs (e.g., corresponding ethyl derivatives) are also highly potent.These agents are essentially active per se since they form electrophiles in presence of water or othernucleophiles. Some of these compounds may decompose explosively if stored for long times at roomtemperature.*N-Methyl-N/-nitro-N-nitrosoguanidine (MNNG)*N-Methyl-N-nitrosourea*N-Methyl-N-nitrosourethane*Streptozotocin

Nitrosamines (highly potent, light-sensitive)Dimethylnitrosamine (Nitrosodimethylamine) (volatile liquid)Diethylnitrosamine (Nitrosodiethylamine) (volatile liquid)

Polycyclic aromatic hydrocarbonsThese highly potent agents are suspected of being active in humans.Benzo(a)pyrene (3,4-Benzpyrene)3-Methylcholanthrene (20-Methylcholanthrene)7,12-Dimethylbenz(a)anthracene (9,10-Dimethyl-l,2-benzanthracene)

MiscellaneousAflatoxin B1. This is an extremely potent hepatocarcinogen suspected of being active in humans. It islight sensitive and subject to electrostatic scattering during weighing of dry solid. Dissolve in solventand calculate amount from spectral absorption.Mitomycin CUrethan (Ethyl carbamate). This is a low-melting, volatile solid. Weigh it in a closed vessel.



Carcinogens Regulated by State of Wisconsin StatuteBecause of their carcinogenic activities, the use of the following chemicals, at the concentrationsindicated below, is regulated by State of Wisconsin Administrative Code 1910.93. This code requiresmaintenance of inventories, use in a regulated area, and other special conditions.

Acrylonitrile (> 10.0 ppm/15 min)2-Acetylaminofluorene (>1.0% solutions or suspensions)4-Aminobiphenyl (4-aminodiphenyl, p-aminobiphenyl) (> 0.1%)Arsenic, inorganic (>10.0 µgM3/8 hrs)Benzidine (4,4'-diaminobiphenyl) (> 0.1%)Bis(chloromethyl)ether (> 0.1%)4-Dimethylaminoazobenzene (p-dimethylaminoazobenzene, N, N-dimethyl-p-phenylazoaniline) (>1.0%)3,3'Dichlorobenzidine (3,3'-Dichloro-4,4'-diaminobiphenyl) (> 1.0%)Ethylene imine (> 1.0%)4,4'-Methylene-bis (2-chloroaniline) (> 1.0%)Methyl chloromethyl ether (chloromethyl methyl ether) (> 0.1%)α-Naphthylamine (1-naphthylamine, 1-aminonaphthalene) (> 1.0%)β-Naphthylamine (2-naphthylamine, 2-aminonaphthalene) (> 0.1%)4-Nitrobiphenyl (p-nitrobiphenyl) (> 0.1%)β-Propiolactone (hydracrylac acid β-lactone) (> 1.0%)N-Nitrosodimethylamine (dimethylnitrosamine) (> 1.0%)Vinyl chloride (> 5.0 ppm/15 min)



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